Field of the Invention
[0001] The present invention relates to novel endogenous variants of erythropoietin (EPO)
and their use for treatment or prevention of a condition associated with tissue damage
due to cell death (apoptosis, necrosis) and inflammtion, in particular for neuroprotection,
e.g. treatment of acute (for example stroke) and chronic disease (for example ALS)
of the nervous system.
Background of the Invention
[0002] Stroke is a debilitating disease which affects more than 400,000 persons per year
in the United States and is the third most common cause of death in the United States.
In addition one-half of neurology inpatients have stroke related problems. At current
trends, this number is projected to jump to one million per year by the year 2050.
When the direct costs (care and treatment) and the indirect costs (lost productivity)
of strokes are considered together, strokes put a burden of $43.3 billion per year
on the society of the United States alone. About 1/3 of patients die in the first
three months, 1/3 remain with severe disabilities, and only 1/3 recover with acceptable
outcome. In 1990 cerebrovascular diseases were the second leading cause of death worldwide,
killing over 4.3 million people world wide. Thus, from a public health perspective,
stroke is one of the most relevant diseases.
[0003] Stroke is characterized by the sudden loss of circulation to an area of the brain,
resulting in a corresponding loss of neurologic function. Also called cerebrovascular
accident or stroke syndrome, stroke is a nonspecific term encompassing a heterogeneous
group of pathophysiologic causes, including thrombosis, embolism, and hemorrhage.
Strokes currently are classified as either hemorrhagic or ischemic. Acute ischemic
stroke refers to strokes caused by thrombosis or embolism and account for 80% of all
strokes.
[0004] Ischemic strokes result from blockage of the arteries that supply the brain, most
commonly in the branches of the internal carotid arteries. The blockage usually results
when a piece of a blood clot (thrombus) or of a fatty deposit (atheroma) due to atherosclerosis
breaks off (becoming an embolus), travels through the bloodstream, and lodges in an
artery that supplies the brain. Blood clots may form when a fatty deposit in the wall
of an artery ruptures. The rupture of such a fatty deposit may also form when a large
fatty deposit slows blood flow, reducing it to a trickle. Blood that flows slowly
is more likely to clot. Thus, the risk of a clot forming in and blocking a narrowed
artery is high. Blood clots may also form in other areas, such as in the heart or
on a heart valve. Strokes due to such blood clots are most common among people who
have recently had heart surgery and people who have a heart valve disorder or an abnormal
heart rhythm (arrhythmia), especially atrial fibrillation. Also, in certain disorders
such as an excess of red blood cells (polycythemia), the risk of blood clots is increased
because the blood is thickened.
[0005] An ischemic stroke can also result, if the blood flow to the brain is reduced, as
may occur when a person loses a lot of blood or has very low blood pressure. Occasionally,
an ischemic stroke occurs when blood flow to the brain is normal but the blood does
not contain enough oxygen. Disorders that reduce the oxygen content of blood include
severe anemia (a deficiency of red blood cells), suffocation, and carbon monoxide
poisoning. Usually, brain damage in such cases is widespread (diffuse), and coma results.
An ischemic stroke can occur, if inflammation or infection narrows blood vessels that
supply the brain. Similarly, drugs such as cocaine and amphetamines can cause spasm
of the arteries, which can lead to a narrowing of the arteries supplying the brain
to such an extent that a stroke is caused.
[0006] The brain requires glucose and oxygen to maintain neuronal metabolism and function.
The inadequate delivery of oxygen to the brain leads to a hypoxia and ischemia results
from insufficient cerebral blood flow. The consequences of cerebral ischemia depend
on the degree and the duration of reduced cerebral blood flow. Neurons can tolerate
ischemia for 30-60 minutes. If flow is not re-established to the ischemic area, a
series of metabolic processes ensue. The neurons become depleted of ATP and switch
over to anaerobic glycolysis, a much less efficient pathway. Lactate accumulates and
the intracellular pH decreases. Without an adequate supply of ATP, ion pumps in the
plasma membrane fail. The resulting influx of sodium, water, and calcium into the
cell causes rapid swelling of neurons and glial cells. Membrane depolarization also
stimulates the massive release of the amino acids glutamate and aspartate, both of
which act as excitatory neurotransmitters in the brain. Glutamate further activates
sodium and calcium ion channels in the neuronal cell membrane namely the well characterized
N-methyl-D-aspartate (NMDA) calcium channel. Excessive calcium influx causes the disordered
activation of a wide range of enzyme systems (proteases, lipases, and nucleases).
These enzymes and their metabolic products, such as oxygen free radicals, damage cell
membranes, genetic material, and structural proteins in the neurons, ultimately leading
to the cell death of neurons (
Dirnagl, U. et al. (1999) Trends Neurosci. 22: 391-397).
[0007] Strokes begin suddenly, develop rapidly, and cause death of brain tissue within minutes
to days. In the ischemic brain, we commonly distinguish two tissue volumes - the core
of the infarction and the surrounding zone, known as ischemic penumbra - the underperfused
and metabolically compromised margin surrounding the irrevocably damaged core. Core
and penumbra are characterized by two different kinds of cell death: necrosis and
apoptosis (which is also called programmed cell death or delayed neuronal cell death).
The severe perfusion deficit in the core causes a breakdown of metabolic processes,
cellular energy supply and ion homeostasis, which causes the cells to lose their integrity
within minutes. Thus, acute necrosis of cell and tissue prevails in the core. In the
penumbra, some residual perfusion is maintained by collateral vessels, which may be
unable to maintain the full functional metabolism, but prevents immediate structural
disintegration. However, over time (hours to several days), the alteration of cellular
homeostasis causes more and more cells to die, and the volume of the infarction increases.
The penumbra has thus to be considered as tissue at risk during the maturation of
the infarct. In this region, apoptosis and inflammatory signaling cascades play an
important role. It may initially constitute 50% of the volume that will end up as
infarction. The mechanisms that lead to delayed cell death provide targets for a specific
neuroprotective therapy in brain regions challenged by ischemia, but which are still
viable.
[0008] Therapeutic options so far are highly disappointing: Thrombolysis with rtPA, the
only therapy with proven efficacy in a major clinical trial (NINDS), is only effective
within a three hour time window, limiting its application to only a few percent of
patients with ischemic stroke. In other words, besides basic supportive therapy, at
present more than 95 % of strokes cannot be treated specifically. This is in sharp
contrast to our knowledge concerning the basic pathophysiology of this disease, which
has emerged over the last decade. In particular, extensive knowledge has accumulated
on mechanisms of parenchymal brain damage and endogenous neuroprotection, as well
as functional and structural reorganization.
[0009] Recently, attention has focused on potential therapeutic roles for endogenous brain
proteins possessing neuroprotective properties. EPO, a glycoprotein hormone produced
primarily by cells of the peritubular capillary endothelium of the kidney, which is
a member of the growth hormone/prolacton cytokine family (
Zhu Y. and D'Andrea A.D: (1994) Curr. Opin. Hematol. 1: 113-118) is a promising candidate. Although EPO was first characterized and is now widely
known for its role as a haematopoietic hormone the detection of EPO and its receptor
(EPOR) in rodent and human brain tissue as well as in cultured neurons and astrocytes
expanded the search for other biological roles of EPO.
[0010] In the brain, a paracrine EPO/(Epo-R)
2 system exists independent of the endocrine system of adult erythropoiesis; neurones
express (Epo-R)
2 and astrocytes produce EPO (
Ruscher et al. (2002) J. Neurosci. 22, 10291-301;
Prass et al. (2003) Stroke 34,1981-1986). It was demonstrated
in vitro and
in vivo that EPO is a potent inhibitor of neuronal apoptosis induced by ischemia and hypoxia
(
Ruscher et al. (2002) J. Neurosci. 22, 10291-301;
Bernaudin, M., et al. (1999) J Cereb Blood Flow Metab. 19: 643-51;
Morishita, E., et al. (1997) Neuroscience. 76: 105-16). It was reported by several groups that addition of EPO to neuronal cultures protects
against hypoxic and glutamic acid toxicity (
Henn F.A: and Braus D.F. (1999) Eur. Arch. Psychiatry Clin. Neurosci. 249: 48-56,
Vogeley K. et al. (2000) Am. J. Psychiatry 157: 34-39) and reduces neurologic dysfunction in rodent models of strike (
Brines M.L. et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97: 10526-10531 and
Bernaudin et al. (1999) J. Cereb. Blood Flow Metab. 10: 643-.651). The promising results of these experiments have been corroborated in human studies
wherein it was shown that EPO therapy for acute stroke is safe and might be beneficial
(
Ehrenreich H. et al. (2002) Mol. Medicine 8: 495-505) and
WO 00/35475 A2. These cell and more particular neuroprotective properties of EPO have led to further
research in this area to substantiate these findings in larger trial and the use of
EPO is now proposed in other indication as well including, for example, schizophrenia
(
Ehrenreich H et al. (2004) Molecular Psychiatry 9: 42-54 and
WO 02/20031 A2).
[0011] For the application of EPO to prevent tissue damage the hematopoietic activity is
often not required and might be detrimental if large amounts of EPO are administered
to treat or ameliorate the effects of, hypoxia or ischemia induced tissue damage.
Therefore, attempts have been made to create EPO variants, which only exhibit the
cell protective property but not the hematopoietic properties.
US 2003/0130197 describes peptide mimetics of EPO for the treatment of neurodegenerative disorders,
which bear no sequence homology to naturally occurring EPO or fragments thereof.
US 6,531,121 discloses a asialoerythropoietin which is generated by complete desialylation of
recombinant EPO showed an increased ability to cross the endothelial cell barrier
and had a decreased hematopoietic activity. Carbamylated erythropoietin (CEPO) was
also shown to exhibit a tissue protective effect but no erythropoietic effect (
Leist et al. (2004) Science 305: 239-242 and
WO 2005/025606 A1.
[0012] WO 2004/043382 discloses human erythropoietin polypeptide variants having altered erythropoietic
activity. The disclosed EPO variants contain amino acid differences in two or more
different EPO-modification regions compared to wild-type human EPO sequences. Such
regions include the carbohydrate content region (A30, H32, P87, W88, P90), the aggregation
region (N24, N38, N83), and the EPOR binding region (T44, T48, N147, and L155). In
some of the disclosed variants, the erythropoietic activity was shown to be decreased,
or the time required to reach the maximal level of such activity was extended. However,
there is no teaching or suggestion of any EPO variants whose hematopoietic activity
is essentially removed while the cell or neuroprotective activity is retained. Finally,
it was shown that a 17-mer peptide of EPO inhibited cell death of two neuronal cell
lines, SK-N-MC and NS20Y (
Campana W.M. et al. (1998) Int. J. MoL Medicine 1: 235-241), while at the same time having no hematopoietic activity. However, 1 ng/ml of the
EPO peptide was needed to elicit the same antiapoptotic effect as 100 pg/ml recombinant
EPO (rhEPO) in NS20Y cells and as 400 pg/ml rhEPO in SK-N-MC cells. Given the apparent
molecular weight of rhEPO of about 66.000 g/mol (the calculated molecular weight is
about 33.000 g/mol but does not include the weight of oligosaccharide residues comprised
in rhEPO) and of about 1.900 g/mol of the EPO peptide a concentration of 1,52 pmol/1
and 6,06 pmol/1, respectively, of rhEPO and 1 nmol/1 of the EPO peptide elicited the
same level of a cell protective effect. Consequently, the EPO peptide is between 650-fold
to 165-fold less active than rhEPO in prevention of cell death. It is evident from
this figures that the EPO region comprised in the 17-mer does not play a major role
in the cell protective function of EPO. Therefore, all EPO variants, which have a
decreased hematopoietic activity known in the prior art suffer from the disadvantage
that they are not natural occurring since they have either lost their natural glycosylation
or they are artificial truncations and/or they have vastly diminished cell protective
activity, if compared to rhEPO. Therefore, there is a need in the prior art to provide
an EPO derivative, which is close to the naturally occurring EPO and which has the
same or better tissue protecting activity as rhEPO but less or no hematopoietic, in
particular no erythropoietic activity.
[0013] This problem is solved by the provision of new EPO variants, which were found to
occur naturally in human and mouse tissue (brain, kidney) and which exhibit a cell
protective activity similar or better to rhEPO but which do not exhibit any significant
hematopoietic activity.
Summary of the Invention
[0014] In one aspect the present invention is concerned with an EPO variant encoding polynucleotide
selected from the group consisting of:
- (a) polynucleotides encoding the mature form of the polypeptides termed hs3, h1-4,
h1-5, hs4, h1-1, h2-I, mS, mG3, mG5, m301, mK3, ha, hAma, hAmE, and hA-10 having the
deduced amino acid sequence as shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18,
20, 22, 50, 51, 52, and 53, respectively;
- (b) a polynucleotide encoding the mature form of the polypeptide termed ha-sequence
without leader consisting of the deduced amino acid sequence as shown in SEQ ID NO:
61;
- (c) polynucleotides having the coding sequence, as shown in SEQ ID NOs: 1,3, 5, 7,
9, 11, 13, 15, 17, 19, 21, 55, 56, 57, and 58 encoding at least the mature form of
the polypeptide;
- (d) a polynucleotide consisting of the coding sequence, as shown in SEQ ID NO: 60
encoding the mature form of the polypeptide termed ha-sequence without leader;
- (e) polynucleotide encoding a humanized version of the polypeptides mS, mG3, mG5,
m301 and mK3 consisting of the deduced amino acid sequence as shown in SEQ ID NOs:
14, 16, 18, 20, and 22,
- (f) polynucleotides encoding a polypeptide comprising a fusion of an amino acid sequence
selected from the group of amino acid sequences as shown in SEQ ID NO: 24, 26, 28,
and 30, at the N-terminus of an amino acid sequence selected from the group of amino
acid sequences as shown in SEQ ID NO: 32, 34, 36, and 38, wherein said fusion has
cell protective and in particular neuroprotective activity but essentially no hematopoietic
activity;
- (g) polynucleotides comprising a fusion of polynucleotide sequences selected from
the group of polynucleotide sequences as shown in SEQ ID NO 23, 25, 27, and 29, 5'
of a polynucleotide sequence selected from the group of polynucleotide sequences as
shown in SEQ ID NO: 31, 33, 35, and 37, wherein said fusion has cell protective and
in particular neuroprotective activity but essentially no hematopoietic activity;
- (h) polynucleotides encoding a derivative of a polypeptide encoded by a polynucleotide
of any one of (a) to (g), wherein in said derivative between 1 and 10 amino acid residues
are conservatively substituted compared to said polypeptide, and said derivative has
cell protective and in particular neuroprotective activity but essentially no hematopoietic
activity;
- (i) polynucleotides encoding a fragment of n polypeptide encoded by a polynucleotide
of any one of (a) to (h), wherein in said fragment between 1 and 10 amino acid residues
are N-and/or C-terminally deleted and/or between 1 and 10 amino acids are deleted
N- and/or C-terminally of the junction compared to said polypeptide, and said fragment
has cell protective and in particular neuroprotective activity but essentially no
hematopoietic activity;
- (j) polynucleotides which are at least 95% identical to a polynucleotide as defined
in any of (a) to (d) and which at the same time have cell protective and in particular
neuroprotective activity but essentially no hematopoietic activity;
- (k) polynucleotides the complementary strand of which hybridizes under stringent conditions
to a polynucleotide as defined in any one of (a) to (j) and which code for a polypeptide
having cell protective and in particular neuroprotective activity but essentially
no hematopoietic activity;
- (l) polynucleotides encoding an EPO variant polypeptide, which comprises the N-terminal
part of full length EPO including helix A and which lacks at least one of the following:
- (i) a fragment of at least 10 amino acids between helix A and helix B;
- (ii) a fragment of at least 10 amino acids of helix B;
- (iii) a fragment of at least 6 amino acids between helix B and helix C;
- (iv) a fragment of at least 10 amino acids of helix C;
- (v) a fragment of at least 20 amino acids between helix C and D; and/or
- (vi) a fragment of at least 10 amino acids of helix D;
wherein said variant has cell protective and in particular neuroprotective activity
but essentially no hematopoietic activity.
- (m) polynucleotides encoding a derivative of a polypeptide encoded by a polynucleotide
of any one of (I), wherein in said derivative between 1 and 10 amino acid residues
are conservatively substituted compared to said polypeptide, and said derivative has
cell protective and in particular neuroprotective activity but essentially no hematopoietic
activity;
- (n) polynucleotides the complementary strand of which hybridizes under stringent conditions
to a polynucleotide as defined in any one of (l) to (m) and which code for a polypeptide
having cell protective and in particular neuroprotective activity but essentially
no hematopoietic activity;
or the complementary strand of such a polynucleotide.
[0015] A further aspect of the present invention is a homolog of an erythropoietin (EPO)
variant encoding polynucleotides from another higher eukaryotic species.
[0016] In a preferred aspect the polynucleotide of the present invention which is DNA, genomic
DNA or RNA.
[0017] In another aspect the present invention is concerned with a vector containing the
polynucleotide of the present invention. It is preferred that the polynucleotide contained
in the vector is operatively linked to expression control sequences allowing expression
in prokaryotic and/or eukaryotic host cells.
[0018] Another aspect of the invention is a host cell genetically engineered with the polynucleotide
of the present invention or the vector of the present invention.
[0019] Another aspect of the invention is a transgenic non-human mammal selected from the
group of non-human primate, horse, bovine, sheep, goat, pig, dog, cat, rabbit, mouse,
rat, guinea pig, hamster and gerbil containing a polynucleotide of the present invention,
a vector of the present invention and/or a host cell of the present invention.
[0020] Another aspect of the invention is a process for producing an EPO variant polypeptide
en-coded by the polynucleotide of the present invention comprising: culturing the
host cell of the present invention and recovering the polypeptide encoded by said
polynucleotide.
[0021] In a preferred embodiment the process of the present invention, further comprises
the step of modifying said EPO variant, wherein the modification is selected from
the group consisting of oxidation, sulfation, phosphorylation, addition of oligosaccharides
or combinations thereof.
[0022] Another aspect of the invention is a process for producing cells capable of expressing
at least one of the EPO variants comprising genetically engineering cells in vitro
with the vector of the invention, wherein said EPO variant polypeptide(s) is(are)
encoded by a polynucleotide of the invention.
[0023] Another aspect of the invention is a polypeptide having the amino acid sequence encoded
by a polynucleotide of the present invention or obtainable by the process of the present
invention.
[0024] Disclosed in the context of the invention is an antibody specifically binding to
the polypeptide of the present invention.
[0025] Another aspect of the invention is a pharmaceutical composition comprising a polynucleotide
of the present invention, a vector of the present invention, a host cell of the present
invention, a polypeptide of the present invention and/or an antibody of the present
invention and a one or more pharmaceutically acceptable carrier.
[0026] Another aspect of the invention is the use of a polynucleotide of the present invention,
a vector of the present invention, a host cell of the present invention, a polypeptide
of the present invention for the manufacture of a medicament for the treatment or
prevention of a condition associated with tissue damage due to cell death, e.g. apoptosis
and necrosis as well as by inflammation.
[0027] In a preferred use of the present invention cell death is induced by ischemia, hypoxia,
bacterial infection, viral infection, autoimmunologically, traumatically, chemically
(e.g. metabolically, toxically) induced, or radiation induced.
[0028] In a preferred use of the present invention the condition is an acute neurodegenerative/neuroinflammatory
disorder or a chronic neurodegenerative/neuroinflammatory disorder, is an acute or
chronic disorder of the heart (e.g. myocardial infarction), lung (e.g. asthma, chronic
obstructive lung disease), kidney (e.g. glomerulonephritis), liver (e.g. chronic liver
failure) or pancreas (e.g. pancreatitis) or said condition is associated with an organ
(e.g. kidney or liver) or cell transplantation (e.g. stem cell)..
[0029] Preferably the acute neurodegenerative and/or neuroinflammatory disorder is selected
from the group consisting of cerebral ischemia or infarction including embolic occlusion
and thrombotic occlusion, reperfusion following acute ischemia, perinatal hypoxic-ischemic
injury, cardiac arrest, intracranial hemorrhage, subarachnoidal hemorrhage and intracranial
lesions (e.g. CNS trauma), spinal cord lesions, intravertebral lesions, whiplash shaken
infant syndrome, infectious encephalitis (e.g. herpes encephalitis), meningitis (e.g.
bacterial), headache (e.g. migraine).
[0030] Preferably the chronic neurodegenerative/neuroinflammatory disorder is selected from
the group consisting of dementias (e.g. Alzheimer's disease, vascular dementias),
Pick's disease, diffuse Lewy body disease, progressive supranuclear palsy (Steel-Richardson
syndrome), multiple sclerosis, multiple system atrophy (including Shy-Drager syndrome),
chronic epileptic conditions associated with neurodegeneration, motor neuron diseases,
degenerative ataxias, cortical basal degeneration, ALS-Parkinson's Dementia complex
of Guam, subacute sclerosing panencephalitis, Huntington's disease, Parkinson's disease,
synucleinopathies, primary progressive aphasia, striatonigral degeneration, Machado-Joseph
disease/spinocerebellar ataxia type 3 and olivopontocerebellar degenerations, Gilles
De La Tourette's disease, bulbar and pseudobulbar palsy, spinal and spinobulbar muscular
atrophy (Kennedy's disease), primary lateral sclerosis, familial spastic paraplegia,
Werdnig-Hoffmann disease, KugelbergWelander disease, Tay-Sach's disease, Sandhoff
disease, familial spastic disease, spastic paraparesis, progressive multifocal leukoencephalopathy,
familial dysautonomia (Riley-Day syndrome), polyneuropathies (e.g. diabetic, alcohol-toxic,
Guillain-Barre-Syndrome, chronic inflammatory demyelinating polyneuropathy), prion
diseases, addiction, affective disorders (e.g. depression), schizophrenic disorders,
chronic fatique syndrome, chronic pain (e.g. lower back pain).
[0031] In a preferred use of the present invention the condition is aging.
[0032] In a preferred use of the present invention the medicament is administered prior
to or after the onset of said condition.
Detailed Description of the Invention
[0033] Unless otherwise defined, all technical and scientific terms used herein have the
same meaning as commonly understood by one of ordinary skill in the art to which this
invention pertains. In case of conflict, the present document, including definitions,
will control. Preferred methods and materials are described below, although methods
and materials similar or equivalent to those described herein can be used in the practice
or testing of the present invention. All publications, patent applications, patents
and other references mentioned herein are incorporated by reference in their entirety.
The materials, methods, and examples disclosed herein are illustrative only and not
intended to be limiting.
[0034] The present invention is based on the surprising observation that EPO variants are
expressed in neuronal tissue and the determination that the variants protected neurons
from damage induced by oxygen and glucose deprivation but did not show hematopoietic
activity. This behavior makes them suitable for use as therapeutics in situations
where the hematopoietic function of EPO is not required or deleterious. Accordingly
a first aspect of the present invention is an EPO variant encoding polynucleotide
selected from the group consisting of:
- (a) polynucleotides encoding the mature form of the polypeptides termed hs3, h1-4,
h1-5, hs4, h1-1, h2-1 mS, mG3, mG5, m301 and mK3 having the deduced amino acid sequence
as shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22, respectively;
- (b) polynucleotides having the coding sequence, as shown in SEQ ID NOs: 1, 3, 5, 7,
9, 11, 13, 15, 17, 19, and 21 encoding at least the mature form of the polypeptide;
- (c) polynucleotide encoding a humanized version of the polypeptides mS, mG3, mG5,
m301 and mK3 consisting of the deduced amino acid sequence as shown in SEQ ID NOs:
14, 16, 18, 20, and 22,
- (d) polynucleotides encoding a polypeptide comprising a fusion of an amino acid sequence
selected from the group of amino acid sequences as shown in SEQ ID NO 24, 26, 28,
and 30, at the N-terminus, preferably directly, i.e. without any intervening amino
acids, of an amino acid sequence selected from the group of amino acid sequences as
shown in SEQ ID NO 32, 34, 36, and 38, wherein said fusion has cell protective and
in particular neuroprotective activity but essentially no hemapoietic activity;
- (e) polynucleotides comprising a fusion of polynucleotide sequences selected from
the group of polynucleotide sequences as shown in SEQ ID NO 23, 25, 27, and 29, 5',
preferably directly 5', i.e. without any intervening polynucleotides, of a polynucleotide
sequence selected from the group of polynucleotide sequences as shown in SEQ ID NO
31, 33, 35, and 37;
- (f) polynucleotides encoding a derivative of a polypeptide encoded by a polynucleotide
of any one of (a) to (e), wherein in said derivative between 1 and 10 amino acid residues
are conservatively substituted compared to said polypeptide, and said derivative has
cell protective and in particular neuroprotective activity but essentially no hematopoietic
activity;
- (g) polynucleotides encoding a fragment of a polypeptide encoded by a polynucleotide
of any one of (a) to (f), wherein in said fragment between 1 and 10 amino acid residues
are N- and/or C-terminally deleted and/or between 1 and 10 amino acids are deleted
N- and or C-terminally of the junction compared to said polypeptide, and said fragment
has cell protective and in particular neuroprotective activity but essentially no
hematopoietic activity;
- (h) polynucleotides which are at least 95% identical to a polynucleotide as defined
in any one of (a) to (b) and which at the same time have cell protective and in particular
neuroprotective activity but essentially no hematopoietic activity; and
- (i) polynucleotides the complementary strand of which hybridizes under stringent conditions
to a polynucleotide as defined in any one of (a) to (h) and which code for a polypeptide
having cell protective and in particular neuroprotective activity but essentially
no hematopoietic activity;
or the complementary strand of such a polynucleotide
[0035] The invention further relates to an EPO variant encoding polynucleotide selected
from the group consisting of:
- (a) polynucleotides encoding the polypeptides termed ha, hAma, hAmE, hA-10 and ha-sequence
without leader, having the deduced amino acid sequence as shown in SEQ ID NOs: 50,
51, 52, 53 and 61 respectively;
- (b) polynucleotides having the coding sequence, as shown in SEQ ID NOs: 55, 56, 57,
58 and 61 encoding at least the mature form of the polypeptide;
- (c) polynucleotides encoding a derivative of a polypeptide encoded by a polynucleotide
of any one of (a) to (b), wherein in said derivative between 1 and 10 amino acid residues
are conservatively substituted compared to said polypeptide, and said derivative has
cell protective and in particular neuroprotective activity but essentially no hematopoietic
activity;
- (d) polynucleotides encoding a fragment of a polypeptide encoded by a polynucleotide
of any one of (a) to (b), wherein in said fragment between 1 and 10 amino acid residues
are N- and/or C-terminally deleted and/or between 1 and 10 amino acids are deleted
N- and or C-terminally of the junction compared to said polypeptide, and said fragment
has cell protective and in particular neuroprotective activity but essentially no
hematopoietic activity;
- (e) polynucleotides which are at least 95% identical to a polynucleotide as defined
in any one of (a) to (b) and which at the same time have cell protective and in particular
neuroprotective activity but essentially no hematopoietic activity; and
- (f) polynucleotides the complementary strand of which hybridizes under stringent conditions
to a polynucleotide as defined in any one of (a) to (e) and which code for a polypeptide
having cell protective and in particular neuroprotective activity but essentially
no hematopoietic activity;
or the complementary strand of such a polynucleotide.
[0036] In a further aspect the polynucleotides of the present invention comprise homologs
of the EPO variants of the present invention derived from another higher eukaryotic
species, in particular from mammals, more preferably from non-human primates; from
rodents, e.g. rat, or guinea pig; ruminant, e.g. cow; or sheep; horse; pig; rabbit;
dog; or cat, which have cell protective and in particular neuroprotective activity
but essentially no hematopoietic activity. In this context the term homolog refers
to a polynucleotide encoding a EPO variant derived from another species, which comprises
essentially the same deletion as the polynucleotides according to SEQ ID NO 1, 3,
5, 7, 9, 11, 13,1 15, 17, 19, 21, 55, 56, 57, 58 or 60. A deletion of a polynucleotide
is considered to be essentially the same, if it involves the deletion of polynucleotides
encoding a polypeptide, which is homologous to the respectively deleted polypeptides
in the EPO variant polypeptides according to SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22 50, 51, 52, 53 or 61. The criterions for determining homology between two
peptide sequences are well established. For this purpose programs as BLASTP can be
used. A deletion is still considered to be essentially the same if it involves 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 55, 56, 57, 58 or 61 more or less nucleotides as the respective
deletion in SEQ ID NO 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21, which are also depicted
in Fig. 2 and 3.
[0037] A further aspect of the present invention is an EPO variant encoding polynucleotide
selected from the group consisting of:
- (a) polynucleotides encoding an EPO variant polypeptide, which comprises the N-terminal
part of full length EPO including helix A and which lacks at least one of the following:
- (i) a fragment of at least 10 amino acids, preferably 11, 12, 13, 14, 15, 16, 17,
18, 19 or 20 amino acids between helix A and helix B;
- (ii) a fragment of at least 10 amino acids, preferably 11, 12, 13, 14, 15, 16, 17,
18, 19,20, 21, 22, 23, 24, 25, 26, 27 or 28 amino acids of helix B;
- (iii) a fragment of at least 6 amino acids between helix B and helix C;
- (iv) a fragment of at least 10 amino acids, preferably 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21 , 22, or 23 amino acids of helix C;
- (v) a fragment of at least 20 amino acids, preferably 21, 22, 23, 24, 25, 26, or 27
amino acids between helix C and D; and/or
- (vi) a fragment of at least 10 amino acids, preferably 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, or 22 of helix D;
wherein said variant has cell protective and in particular neuroprotective activity
but essentially no hematopoietic activity.
- (b) polynucleotides encoding a derivative of a polypeptide encoded by a polynucleotide
of any one of (a), wherein in said derivative between 1 and 10 amino acid residues
are conservatively substituted compared to said polypeptide, and said derivative has
cell protective and in particular neuroprotective activity but essentially no hematopoietic
activity;
- (c) polynucleotides the complementary strand of which hybridizes under stringent conditions
to a polynucleotide as defined in any one of (a) to (b) and which code for a polypeptide
having cell protective and in particular neuroprotective activity but essentially
no hematopoietic activity;
or the complementary strand of such a polynucleotide.
[0038] In this context helix A, B, C, and D of the EPO polypeptide are regions homologous
to the respective helix A, B, C, and D regions of full length EPO from mouse and human
as outlined in Fig. 4. It is well known in the art how to determine homologies between
two polypeptide sequences and someone of skill in the art will be capable to align
a given EPO polypeptide sequence derived, e.g. from another species, and to determine
the respective position of helix A, B, C, and D in this EPO polypeptide. It is preferred
that the EPO variant polynucleotide is derived from a higher eukaryote, in particular
a mammal or bird. Preferred mammals are humans, non-human primates; rodents, e.g.
rat, or guinea pig; ruminant, e.g. cow; or sheep; horse; pig; rabbit; dog; or cat.
A larger number of such full length EPO encoding polynucleotides from various species
are known, including without limitation cat (Gene Bank Acc. L10606), pig (Gene Bank
Acc. 10607), sheep (Gene Bank Acc.10610), dog (Gene Bank Acc. L13027), macaque (Gene
Bank Acc. M18189), rhesus monkey (Gene Bank Acc. L10609), mouse (Gene Bank Acc. 12930),
rat (Gene Bank Acc. L10608), human (Gene Bank Acc. M11319) Bos taurus (Gene Bank Acc.
U44762) and Bos indicus (Gene Bank Acc. L41354).
[0039] Preferably the polynucleotides encoding an EPO variant polypeptide lacks the following:
(i); (ii); (iii); (iv); (v); (vi); (i) and (ii); (i) and (iii); (i) and (iv); (i)
and (v); (i) and (vi); (ii) and (iii); (ii) and (iv); (ii) and (v); (ii) and (vi);
(iii) and (iv); (iii) and (v); (iii) and (vi); (iv) and (v); (iv) and (vi); (v) and
(vi); (i), (ii) and (iii); (i), (ii) and (iv), (i), (ii) and (v), (i), (ii), (vi),
(i), (iii) and (iv); (i), (iii) and (v); (i), (iii) and (vi); (i), (iv) and (v); (i),
(iv) and (vi); (i), (v) and (vi); (ii), (iii) and (iv); (ii), (iii) and (v); (ii),
(iii) and (vi); (ii), (iv) and (v); (ii), (iv) and (vi); (ii), (v) and (vi); (iii),
(iv) and (v); (iii), (iv) and (vi); (iii), (v) and (vi); or (iv), (v) and (vi).
[0040] A polypeptide that exhibits cell protective activity is a polypeptide that has at
least 50% (e.g., at least: 55%; 60%; 65%; 70%; 75%; 80%; 85%; 90%; 95%; 98%; 99%;
99.5%; or 100% or even more) of the ability of the respective EPO variant to protect
neurons from damage by apoptosis, wherein the apoptosis is induced by oxygen or glucose
deprivation, by chemical or radiation exposure or by viral or bacterial infection.
Assays to determine damage to cells, in particular to neuronal cells are known in
the art. A suitable assays is the oxygen glucose deprivation assay described herein
below. In the described assay the readout is the amount of lactate dehydrogenase activity
(LDH). However, a variety of other methods exist, which allow assessing the damage
induced in a cell and in particular the amount of cell death (e.g. apoptosis, necoris).
These assays include without limitation Tunnel assays, MTT-assay, life/death assay
by staining (e.g. Ethidium bromide and acridine orange staining), caspase assay, electron
microscopy, DNA-laddering, which are all well known in the art.
[0041] Am EPO variant polypeptide that exhibits essentially no hematopoietic activity is
a polypeptide, which elicits in art known colony formation assays, an example of which
is described below, at the same molar concentration as the rhEPO and wt mEPO, respectively,
less than 10% of the CFU-E (Colony forming unit-erythroblast), preferably less than
9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. The respective CFU-E numbers are calculated
for a given rhEPO, wt mEPO or EPO variant by subtracting from each value the number
of CFU-E observed in a control reaction (without wt or EPO variant).
[0042] In the context of the polypeptides of the present invention the term "junction" refers
to the site wherein two amino acids follow each other which are not consecutive in
the rhEPO or mouse wt EPO and which are potentially the result of splice events or
other rearrangements in the EPO mRNA. The respective junction of the EPO variants
of the present invention can be derived from Fig. 4, e.g. is ENIT | VGQQ for hS3,
VGQQ | ALLV for h1-4, VNFY | ALL for h1-5, KRME|PWEP for hS4, ITVP|GPVG for h1-1,
LNEN|NHC for h2-1, KRME|KELM for mS, LLAN|FLRG for mG3, DTFC|RRGD for mG5, KVNF|LRGK
for m301 or LSEA|VHGR for mK3.
[0043] The polynucleotide molecules of the invention can be synthesized
in vitro (for example, by phosphoramidite-based synthesis) or can be obtained from a cell,
such as the cell of a mammal.
[0044] The EPO variants termed mS, mG3, mG5, m301 and mK3 having the deduced amino acid
sequence as shown in SEQ ID NOs 14, 16, 18, 20, and 22, respectively were isolated
from mouse The mouse sequence is highly homologous to the human sequence. An alignment
of the amino acid sequences of EPO derived from humans and mouse is provided in Fig.
4. As is apparent the mouse sequence is distinguished from the human sequence by the
lack of an alanine residue at position 8 and by the following 39 substitutions (the
numbering is according to the respective amino acid position in the human EPO, the
first amino acid indicated is the human amino acid at that position and the second
is the corresponding mouse amino acid):
4H →
4P,
6C →
6R,
9W →
9T;
11W →
11L,
18S →
18L;
19L →
19I;
27G →
27C;
43L →
43I,
52I →
52V;
54T →
54M;
60H →
60G;
61C→
61P;
62S →
62R;
64N →
64S;
84G →
84E;
85Q →
85E;
95A →
95S;
101V →
101I;
103R →
103Q;
104G →
104A;
109V →
109A;
115W →
115P;
117P →
117T;
122V →
122I;
126V →
126I;
134T →
134S;
138A →
138V;
145A →
145L;
146I →
146M;
151A →
151T;
152A →
152T;
153S →
153P;
154A →
154P;
160I →
160L;
162A →
162V;
166R →
166C;
173S →
173A;
187A →
187V and
190T →
190R. A humanized mS, mG3, mG5, m301 or mK3 carries the additional alanine residue at
position 8 and/or at one or more preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36 or 37 positions the human rather than the mouse amino acid sequence. It is
particularly preferred that mS, mG3, mG5, m301 and mK3 are fully humanized, i.e. that
every amino acid at the above outlined positions, in as far as they are present in
the respective variant, is of the human sequence rather than the mouse sequence. It
is expected that the humanization of the mouse variants will diminish any immunological
problems, which might be encountered when using in the treatment of humans.
[0045] The EPO variant nucleic acid molecules of the invention can be DNA, cDNA, genomic
DNA, synthetic DNA, or, RNA, and can be double-stranded or single-stranded, the sense
and/or an antisense strand. These molecules can be produced by, for example, polymerase
chain reaction (PCR) or generated by treatment with one or more restriction endonucleases.
A ribonucleic acid (RNA) molecule can be produced by
in vitro transcription.
[0046] The polynucleotide molecules of the invention can contain naturally occurring sequences,
or sequences that differ from those that occur naturally, but, due to the degeneracy
of the genetic code, encode the same polypeptide, i.e. the polypeptides with SEQ ID
NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22. In addition, these nucleic acid molecules
are not limited to coding sequences, e.g., they can include some or all of the non-coding
sequences that lie upstream or downstream from a coding sequence.
[0047] In addition, the isolated nucleic acid molecules of the invention can encompass segments
that are not found as such in the natural state. Thus, the invention encompasses recombinant
nucleic acid molecules incorporated into a vector (for example, a plasmid or viral
vector) or into the genome of a heterologous cell (or the genome of a homologous cell,
at a position other than the natural chromosomal location). Recombinant nucleic acid
molecules and uses therefore are discussed further below.
[0048] In preferred embodiments the polynucleotides of the present invention also comprise
nucleic acid molecules which are at least 95% , preferably 96%, 97%, 98%, or 99% identical
to: (a) a nucleic acid molecule that encodes the polypeptide of SEQ ID NO: 2, 4, 6,
8, 10, 12, 14, 16, 18, 20, 22, 50, 51, 52, 53 or 61 and (b) the nucleotide sequence
of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 55, 56, 57, 58 or 60 respectively
and which at the same time cell protective and in particular neuroprotective activity
but essentially no hematopoietic activity
The determination of percent identity between two sequences is accomplished using
the mathematical algorithm of
Karlin and Altschul (1993) Proc. Natl. Acad. ScL USA 90: 5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTP programs of
Altschul et al. (1990) J. Mol. Biol. 215: 403-410. BLAST nucleotide searches are performed with the BLASTN program, score = 100, word
length = 12, to obtain nucleotide sequences homologous to the EPO variant polypeptide
encoding nucleic acids. BLAST protein searches are performed with the BLASTP program,
score = 50, wordlength = 3, to obtain amino acid sequences homologous to the EPO variant
polypeptide, respectively. To obtain gapped alignments for comparative purposes, Gapped
BLAST is utilized as described in
Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST pro-grams, the default parameters of the respective
programs are used.
[0049] Hybridization can also be used as a measure of homology between two nucleic acid
sequences. A nucleic acid sequence encoding any of the EPO variants disclosed herein,
or a derivative or fragment thereof, can be used as a hybridization probe according
to standard hybridization techniques. The hybridization of an EPO variant probe to
DNA or RNA from a test source (e.g., a mammalian cell) is an indication of the presence
of the relevant EPO DNA or RNA in the test source. Hybridization conditions are known
to those skilled in the art and can be found in
Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6, 1991. Stringent conditions are defined as equivalent to hybridization in 6X sodium chloride/sodium
citrate (SSC) at 45°C, followed by a wash in 0.2 X SSC, 0.1 % SDS at 65°C. When selecting
a probe specific for a variant carrying an internal deletion it is preferred that
the probe used to detect homologous nucleic acids overlaps the boundaries of the deletion,
e.g. hs3, hi -4, hi -5, hS4, mS, mG3, mG5 or m301. In cases where the splicing leads
to an alternate C-terminus of the protein, e.g. hi -1, h2-I or mK3 it is preferred
that the probe used to detect homologous DNA sequences overlaps the boundaries between
the known EPO sequence and the alternate C-terminus. For example, a probe could be
designed, which comprises 10 complementary bases 5' of the splice site and 10 complementary
bases 3' of the splice site.
[0050] An "isolated DNA" is either (1) a DNA that contains sequence not identical to that
of any naturally occurring sequence, or (2), in the context of a DNA with a naturally-occurring
sequence (e.g., a cDNA or genomic DNA), a DNA free of at least one of the genes that
flank the gene containing the DNA of interest in the genome of the organism in which
the gene containing the DNA of interest naturally occurs. The term therefore includes
a recombinant DNA incorporated into a vector, into an autonomously replicating plasmid
or virus, or into the genomic DNA of a prokaryote or eukaryote. The term also includes
a separate molecule such as a cDNA where the corresponding genomic DNA has introns
and therefore a different sequence; a genomic fragment that lacks at least one of
the flanking genes; a fragment of cDNA or genomic DNA produced by polymerase chain
reaction (PCR) and that lacks at least one of the flanking genes; a restriction fragment
that lacks at least one of the flanking genes; a DNA encoding a non-naturally occurring
protein such as a fusion protein, mutein, or fragment of a given protein; and a nucleic
acid which is a degenerate variant of a cDNA or a naturally occurring nucleic acid.
In addition, it includes a recombinant nucleotide sequence that is part of a hybrid
gene, i.e., a gene encoding a non-naturally occurring fusion protein. It will be apparent
from the foregoing that isolated DNA does not mean a DNA present among hundreds to
millions of other DNA molecules within, for example, cDNA or genomic DNA libraries
or genomic DNA restriction digests in, for example, a restriction digest reaction
mixture or an electrophoretic gel slice.
[0051] A further aspect of the present invention is a vector containing the polynucleotide(s)
of the present invention or a protein encoded by a polynucleotide of the present invention
The term "vector" refers to a protein or a polynucleotide or a mixture thereof which
is capable of being introduced or of introducing the proteins and/or nucleic acid
comprised into a cell. It is preferred that the proteins encoded by the introduced
polynucleotide are expressed within the cell upon introduction of the vector.
[0052] In a preferred embodiment the vector of the present invention comprises plasmids,
phagemids, phages, cosmids, artificial mammalian chromosomes, knock-out or knock-in
constructs, viruses, in particular adenoviruses, vaccinia viruses, attenuated vaccinia
viruses, canary pox viruses, lentivirus (
Chang, L.J. and Gay, E.E. (20001) Curr. Gene Therap. 1:237-251), herpes viruses, in particular Herpes simplex virus (HSV-1,
Carlezon, W.A. et al. (2000) Crit. Rev. Neurobiol.), baculovirus, retrovirus, adeno-associated-virus (
AAV, Carter, P.J. and Samulski, R.J. (2000) J. Mol. Med. 6:17-27), rhinovirus, human immune deficiency virus (HIV), filovirus and engineered versions
thereof (see, for example,
Cobinger G. P. et al (2001) Nat. Biotechnol. 19:225-30), virosomes, "naked" DNA liposomes, and nucleic acid coated particles, in particular
gold spheres. Particularly preferred are viral vectors like adenoviral vectors or
retroviral vectors (
Lindemann et al. (1997) Mol. Med. 3:466-76 and
Springer et al. (1998) Mol. Cell. 2:549-58). Liposomes are usually small unilamellar or multilamellar vesicles made of cationic,
neutral and/or anionic lipids, for example, by ultrasound treatment of liposomal suspensions.
The DNA can, for example, be ionically bound to the surface of the liposomes or internally
enclosed in the liposome. Suitable lipid mixtures are known in the art and comprise,
for example, DOTMA (1, 2-Dioleyloxpropyl-3-trimethylammoniumbromid) and DPOE (Dioleoylphosphatidyl-ethanolamin)
which both have been used on a variety of cell lines.
[0053] Nucleic acid coated particles are another means for the introduction of nucleic acids
into cells using so called "gene guns", which allow the mechanical introduction of
particles into the cells. Preferably the particles itself are inert, and therefore,
are in a preferred embodiment made out of gold spheres.
[0054] In a further aspect the polynucleotide of the present invention is operatively linked
to expression control sequences allowing expression in prokaryotic and/or eukaryotic
host cells. The transcriptional/translational regulatory elements referred to above
include but are not limited to inducible and non-inducible, constitutive, cell cycle
regulated, metabolically regulated promoters, enhancers, operators, silencers, repressors
and other elements that are known to those skilled in the art and that drive or otherwise
regulate gene expression. Such regulatory elements include but are not limited to
regulatory elements directing constitutive expression like, for example, promoters
transcribed by RNA polymerase III like , e.g., promoters for the snRNA U6 or scRNA
7SK gene, the cytomegalovirus hCMV immediate early gene, the early or late promoters
of SV40 adenovirus, viral promoter and activator sequences derived from, e.g., NBV,
HCV, HSV, HPV, EBV, HTLV, MMTV or HIV; which allow inducible expression like, for
example, CUP-1 promoter, the tet-repressor as employed, for example, in the tet-on
or tet-off systems, the
lac system, the
trp system; regulatory elements directing tissue specific expression, preferably nerve
cell specific expression, e.g. promoter (e.g. Thy-1.2, NSE, , myosin light chain II,
tyrosine hydroxylase, CaMKIIalpha promoter, platelet-derived growth factor beta-chain
(PDGF), dopamine beta-hydroxylase, Tau, regulatory elements (e.g. NRSE/RE-1; neuron-restrictive
silencing element/repressor element 1) directing cell cycle specific expression like,
for example, cdc2, cdc25C or cyclin A; or the
TAC system, the
TRC system, the major operator and promoter regions of phage A, the control regions of
fd coat protein, the promoter for 3-phosphoglycerate kinase, the promoters of acid
phosphatase, and the promoters of the yeast α- or α-mating factors.
[0055] As used herein, "operatively linked" means incorporated into a genetic construct
so that expression control sequences effectively control expression of a coding sequence
of interest.
[0056] Similarly, the polynucleotides of the present invention can form part of a hybrid
gene encoding additional polypeptide sequences, for example, a sequence which encodes
a protein that functions as a marker or reporter. The hybrid gene can lead to a fusion
protein or the two or more parts can be separated by internal ribosomal entry sites
(IRES) sequence, which lead to the expression of two or more separate proteins. Examples
of marker and reporter genes include
β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside
phosphotransferase (neo
r, G418
r), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine
kinase (TK), lacZ (encoding
β-galactosidase), green fluorescent protein (GFP) and variants thereof and xanthine
guanine phosphoribosyltransferase (XGPRT). As with many of the standard procedures
associated with the practice of the invention, skilled artisans will be aware of additional
useful reagents, for example, additional sequences that can serve the function of
a marker or reporter. If the expression of the hybrid gene leads to one polypeptide
the hybrid polypeptide will usually include a first portion and a second portion;
the first portion being a EPO variant polypeptide and the second portion being, for
example, the reporter described above or an Ig constant region or part of an Ig constant
region, e.g., the CH2 and CH3 domains of IgG2a heavy chain. Other hybrids could include
a heterologous peptide sequence to facilitate purification and/or detection, e.g.
an antigenic tag like, for example, a myc tag, or a tag with preferential binding
to a region, e.g. chitin taq or His tag. Recombinant nucleic acid molecules can also
contain a polynucleotide sequence encoding a EPO variant polypeptide operatively linked
to a heterologous signal sequence. Such signal sequences can direct the protein to
different compartments within the cell and are well known to someone of skill in the
art. A preferred signal sequence is a sequence that facilitates secretion of the resulting
protein. Preferably these signal and/or taq sequences are designed in such that they
can be cleaved of the EPO variant after purification to provide an essentially pure
protein without two many amino acids, preferably not more than 10 additional amino
acids to the final EPO. Such cleavage sites are well known in the art and comprise,
e.g endopeptidase cleavage sites and intein cleavage sites.
[0057] Another aspect of the present invention is a host cell genetically engineered with
the polynucleotide or the vector as outlined above. The host cells that may be used
for purposes of the invention include but are not limited to prokaryotic cells such
as bacteria (for example, E.
coli and
B. subtilis)
, which can be transformed with, for example, recombinant bacteriophage DNA, plasmid
DNA, or cosmid DNA expression vectors containing the polynucleotide molecules of the
invention; simple eukaryotic cells like yeast (for example,
Saccharomyces and
Pichia)
, which can be transformed with, for example, recombinant yeast expression vectors
containing the polynucleotide molecule of the invention; insect cell systems like,
for example, Sf9 of Hi5 cells, which can be infected with, for example, recombinant
virus expression vectors (for example, baculovirus) containing the polynucleotide
molecules of the invention; Xenopus oocytes, which can be injected with, for example,
plasmids; plant cell systems, which can be infected with, for example, recombinant
virus expression vectors (for example, cauliflower mosaic virus (CaMV) or tobacco
mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (for
example, Ti plasmid) containing a EPO variant nucleotide sequence; or mammalian cell
systems (for example, COS, CHO, BHK, HEK293, VERO, HeLa, MDCK, Wi38, Swiss 3T3 and
NIH 3T3 cells), which can be transformed with recombinant expression constructs containing,
for example, promoters derived, for example, from the genome of mammalian cells (for
example, the metallothionein promoter) from mammalian viruses (for example, the adenovirus
late promoter, CMV IE and the vaccinia virus 7.5K promoter) or from bacterial cells
(for example, the tet-repressor binding is employed in the tet-on and tet-off systems).
Also useful as host cells are primary or secondary cells obtained directly from a
mammal and transfected with a plasmid vector or infected with a viral vector. Depending
on the host cell and the respective vector used to introduce the polynucleotide of
the invention the polynucleotide can integrate, for example, into the chromosome or
the mitochondrial DNA or can be maintained extrachromosomally like, for example, episomally
or can be only transiently comprised in the cells.
[0058] Since EPO is heavily glycosylated in vivo it is desirable to choose an expression
system, which provides faithful glycosylation of the protein. Consequently, it is
preferred to introduce the polynucleotides encoding the EPO slice variants of the
present invention into higher eukaryotic cells, in particular into mammalian cells,
e.g. COS, CHO, BHK, HEK293, VERO, HeLa, MDCK, Wi38, Swiss 3T3 or NIH 3T3 cells.
[0059] A further aspect of the present invention is a transgenic non-human mammal containing
a polynucleotide, a vector and/or a host cell as described above. The animal can be
a mosaic animal, which means that only part of the cells making up the body comprise
polynucleotides, vectors, and/or cells of the present invention or the mammal can
be a transgenic animal which means that all cells of the animal comprise the polynucleotides
and/or vectors of the present invention or are derived from a cell of the present
invention. Mosaic or transgenic animals can be either homo- or heterozygous with respect
to the polynucleotides of the present invention contained in the cell. In a preferred
embodiment the transgenic animals are either homo- or heterozygous knock-out or knock-in
animals with respect to the genes which code for the proteins of the present invention.
The mammal is selected from the group of non-human primate horse, bovine, sheep, goat,
pig, dog, cat, goat, rabbit, mouse, rat, guinea pig, hamster, and gerbil.
[0060] Another aspect of the present invention is a process for producing an EPO variant
polypeptide encoded by a polynucleotide of the present invention comprising: culturing
the host cell described above and recovering the polypeptide encoded by said polynucleotide.
Preferred combinations of host cells and vectors are outlined above and further combination
will be readily apparent to someone of skill in the art. Depending on the intended
later use of the recovered peptide a suitable cell type can be chosen. As outlined
above eukaryotic cells are preferably chosen, if it is desired that the proteins produced
by the cells exhibit an essentially natural pattern of glycosylation and prokaryotic
cells are chosen, if, for example, glycosylation or other modifications, which are
normally introduced into proteins only in eukaryotic cells, are not desired or not
needed.
[0061] It is known in the prior art that the pharmacokinetic of protein drugs can be significantly
altered by modification of the protein. For full length EPO it has been described
that glycosylation, in particular the presence of sialic acid residues at the end
of the oligosaccharide side chains attributes to the circulation time (
WO 95/05465) and that removal of sialic acid groups exposes galactose residues, which increases
clearance by the liver. Therefore, one approach taken to enhance EPO circulation time
was the increase in sialic acid residues. Several approaches, thus, involve the provision
of additional glycosylation sites (see e.g.
WO 91/05867,
WO 94/09257 and
WO 01/81405. Such modified EPO analogs may have at least one additional N-linked and/or O-linked
carbohydrate chain. Other attempts to improve the half life of EPO involved the addition
of polyethylene glycol residues (PEG) of varying length the amino acid backbone (see
e.g.
WO 00/32772,
WO 01/02017,
WO 03/029291. Another attempt used the modification of EPO molecules with at least one N-linked
and/or O-linked oligosaccharide which were further modified with oxidation, sulfation,
phosphorylation PEGylation or a combination thereof (see
WO 2005/025606). All these approaches can equally be employed to extend the half life of the EPO
variants of the present invention and accordingly in a preferred embodiment above
process further comprising the step of modifying the EPO variant, wherein the modification
is selected from the group consisting of oxidation, sulfation, phosphorylation, addition
of oligosaccharides or combinations thereof. If the addition of further N-linked or
O-linked oligonucleotides is desired it is possible to introduce them by introducing
additional glycosylation sites as has been described in the prior art, e.g. at positions
30, 51, 57, 69, 88, 89, 136 and/or 138, if the respective position is present in the
variant of the present invention (see
WO 01/81405).
[0062] A further aspect of the invention is a process for producing cells capable of expressing
at least one of the EPO variants comprising genetically engineering cells
in vitro with the vector of claim 3 or 4, wherein said EPO variant polypeptide(s) is(are)
encoded by a polynucleotide of the present invention.
[0063] Another aspect of the invention is a polypeptide having the amino acid sequence encoded
by a polynucleotide of the invention or obtainable by the process mentioned above.
The polypeptides of the invention include all those disclosed herein and fragments
of these polypeptides, which carry between 1 and 10 N- and/or C-terminal deletions.
Preferably, the deletions are less than 10, less than 9, less than 8, less than 7,
less than 8, less than 7, less than 6, less than 5, less than 4, less than 3, less
than 2, less than 1 amino acids. The polypeptides embraced by the invention also include
fusion proteins that contain either the EPO slice variant as indicated in SEQ ID Nos
2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 or humanized version of 14, 16, 18, 20 and
22 or a fragment thereof as defined above fused to an unrelated amino acid sequence.
The unrelated sequences can comprise additional functional domains or signal peptides.
Signal peptides are described in greater detail and exemplified below.
[0064] The polypeptides can be any of those described above but with not more than 10 (e.g.,
not more than: 10, nine, eight, seven, six, five, four, three, two, or one) conservative
substitutions. Conservative substitutions are known in the art and typically include
substitution of, e.g. one polar amino acid with another polar amino acid and one acidic
amino acid with another acidic amino acid. Accordingly, conservative substitutions
preferably include substitutions within the following groups of amino acids: glycine,
alanine, valine, proline, isoleucine, and leucine (non polar, aliphatic side chain);
aspartic acid and glutamic acid (negatively charged side chain); asparagine, glutamine,
methionine, cysteine, serine and threonine (polar uncharged side chain); lysine, histidine
and arginine; and phenylalanine, tryptophane and tyrosine (aromatic side chain); and
lysine, arginine an histidine (positively charged side chain). It is well known in
the art how to determine the effect of a given substitution, e.g. on pK
1, etc. All that is required of a polypeptide having one or more conservative substitutions
is that it has at least 50% (e.g., at least: 55%; 60%; 65%, 70%; 75%; 80%; 85%; 90%;
95%; 98%; 99%; 99.5%; or 100% or more) of the ability of the unaltered EPO variant
to protect neurons from damage/cell death (e.g. by apoptosis or necrosis), wherein
the cell death is induced by oxygen and/or glucose deprivation, by toxic, chemical,
physical, mechanical, inflammatory or radiation exposure or by viral or bacterial
infection.
[0065] Both polypeptides and peptides can be produced by standard
in vitro recombinant DNA techniques and
in vivo transgenesis, using nucleotide sequences encoding the appropriate polypeptides or
peptides. Methods well-known to those skilled in the art can be used to construct
expression vectors containing relevant coding sequences and appropriate transcriptional/translational
control signals. See, for example, the techniques described in
Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Ed.) [Cold Spring Harbor
Laboratory, N.Y., 1989], and
Ausubel et al., Current Protocols in Molecular Biology [Green Publishing Associates
and Wiley Interscience, N.Y., 1989].
[0066] Polypeptides and fragments of the invention also include those described above, but
modified for
in vivo use by the addition, at the amino- and/or carboxyl-terminal ends, of blocking agents
to facilitate survival of the relevant polypeptide
in vivo. This can be useful in those situations in which the peptide termini tend to be degraded
by proteases prior to cellular uptake. Such blocking agents can include, without limitation,
additional related or unrelated peptide sequences that can be attached to the amino
and/or carboxyl terminal residues of the peptide to be administered. This can be done
either chemically during the synthesis of the peptide or by recombinant DNA technology
by methods familiar to artisans of average skill.
[0067] Alternatively, blocking agents such as pyroglutamic acid or other molecules known
in the art can be attached to the amino and/or carboxyl terminal residues, or the
amino group at the amino terminus or carboxyl group at the carboxyl terminus can be
replaced with a different moiety. Likewise, the peptides can be covalently or noncovalently
coupled to pharmaceutically acceptable "carrier" proteins prior to administration.
[0068] The term "isolated" polypeptide or peptide fragment as used herein refers to a polypeptide
or a peptide fragment which either has no naturally-occurring counterpart or has been
separated or purified from components which naturally accompany it, e.g., in tissues
such as tongue, pancreas, liver, spleen, ovary, testis, muscle, joint tissue, neural
tissue, gastrointestinal tissue or tumor tissue, or body fluids such as blood, serum,
or urine. Typically, the polypeptide or peptide fragment is considered "isolated"
when it is at least 70%, by dry weight, free from the proteins and other naturally-occurring
organic molecules with which it is naturally associated. Preferably, a preparation
of a polypeptide (or peptide fragment thereof) of the invention is at least 80%, more
preferably at least 90%, and most preferably at least 99%, by dry weight, the polypeptide
(or the peptide fragment thereof), respectively, of the invention. Thus, for example,
a preparation of polypeptide x is at least 80%, more preferably at least 90%, and
most preferably at least 99%, by dry weight, polypeptide x. Since a polypeptide that
is chemically synthesized is, by its nature, separated from the components that naturally
accompany it, the synthetic polypeptide is "isolated."
[0069] An isolated polypeptide (or peptide fragment) of the invention can be obtained, for
example, by extraction from a natural source (e.g., from tissues or bodily fluids);
by expression of a recombinant nucleic acid encoding the polypeptide; or by chemical
synthesis. A polypeptide that is produced in a cellular system different from the
source from which it naturally originates is "isolated," because it will necessarily
be free of components which naturally accompany it. The degree of isolation or purity
can be measured by any appropriate method, e.g., column chromatography, polyacrylamide
gel electrophoresis, or HPLC analysis.
[0070] Disclosed in the context of the invention is an antibody, which specifically binds
to the EPO variant polypeptide encoded by polynucleotides of the invention or obtainable
by the process mentioned above. The term "antibody" comprises monoclonal and polyclonal
antibodies and binding fragments thereof, in particular Fc-fragments as well as so
called "single-chain-antibodies" (
Bird R. E. et al (1988) Science 242:423-6), chimeric, humanized, in particular CDR-grafted antibodies, and dia or tetrabodies
(
Holliger P. et al (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6444-8). Also comprised are immunoglobulin like proteins that are selected through techniques
including, for example, phage display to specifically bind to the polypeptides of
the present invention. In this context the term "specific binding" refers to antibodies
raised against peptides derived from splice junctions or junctions created by other
processes, e.g. ENIT I VGQQ of hS3, VGQQ | ALLV of h1-4, VNFY | ALLV of h1-5, KRME
| PWEP of hS4, ITVP I GPVG of h1-1, LNEN | NHC of h2-1, KRME | KELM of mS, LLAN |
FLRG of mG3, DTFC | RRGD of mG5, KVNF | LRGK of m301 or LSEA | VHGR of mK3. Such peptides
can comprise additional or less N- or C-terminal amino acids. An antibody is considered
to be specific to the EPO variant, if its affinity towards the variant it at least
50-fold higher, preferably 100-fold higher, more preferably at least 1000-fold higher
than towards the full length human or murine EPO. Preferably specific antibodies of
the present invention do not or essentially do not bind to full length human or murine
EPO. It is well known in the art how to make antibodies and to select antibodies with
a given specificity.
[0071] A further aspect of the present invention concerns the use of a polynucleotide, a
vector, a host cell or a polypeptide of the present invention for the manufacture
of a medicament for the treatment or prevention of a condition associated with tissue
damage due to cell death (e.g. apoptosis and necrosis) . The apoptosis or necrosis
leads to the cell destruction, which can be prevented or ameliorated when using the
polynucleotide, vector, host cell or polypeptide of the present invention. Cell death
can be induced by many different internal and external stimuli and include preferably
ischemia, hypoxia, bacterial or viral infection, radiation, or induced by metabolic,
toxic, chemical, autoimmunologic, or traumatic stimuli. It is well known in the art
how to detect cell death like, for example, using morphological criteria, a TUNNEL
assay, MTT-assay, life/death assay by staining (e.g. Ethidium bromide and acridine
orange staining), caspase assay, electron microscopy, DNA-laddering or the LDH release
assay described below. For example, apoptosis is characterized by chromatin fragmentation,
extravasation of cellular contents and eventually death of the cell. It has been recognized
to play a role in many acute or chronic pathologic processes. Accordingly, a preferred
use of the present in vention comprises the administration of polynucleotides, vectors,
host cells or polypeptides of the present invention to prevent, treat or ameliorate
acute and chronic neurodegenerative or neuroinflammatory disorders, acute or chronic
disorder of the heart (e.g. myocardial infarction), lung (e.g. asthma, chronic obstructive
lung disease), kidney (e.g. glomerulonephritis), liver (e.g. chronic liver failure)
or pancreas(e.g. pancreatitis), as well as conditions associated with cell (e.g. stem
cell) or organ transplantation (e.g. kidney or liver). In this respect is also envisioned
that the EPO variants of the present invention can be included in storage solutions
used for storing organs or limbs for transport and/or after traumatic injury.
[0072] Acute neurodegenerative disorders include, but are not limited to, various types
of acute neurodegenerative disorders associated with neuronal cell death including
cerebrovascular insufficiency, focal or diffuse brain trauma, diffuse brain damage,
and spinal cord injury. Examples of acute neurodegenerative disorders are: cerebral
ischemia or infarction including embolic occlusion and thrombotic occlusion, reperfusion
following acute ischemia, perinatal hypoxic-ischemic injury, cardiac arrest, as well
as intracranial hemorrhage of any type (such as epidural, subdural, subarachnoid and
intracerebral), and intracranial and intravertebral lesions (such as contusion, penetration,
shear, compression and laceration), whiplash shaken infant syndrome infectious encephalitis
(e.g. herpes encephalitis), meningitis (e.g. bacterial), headache (e.g. migraine).
[0073] Chronic neurodegenerative disorders that can be treated with the EPO variants of
the present invention include, but are not limited to, dementias (e.g. Alzheimer's
disease, vascular dementias), Pick's disease, diffuse Lewy body disease, progressive
supranuclear palsy (Steel-Richardson syndrome), multiple sclerosis, multiple system
atrophy (including Shy-Drager syndrome), chronic epileptic conditions associated with
neurodegeneration, motor neuron diseases including amyotrophic lateral sclerosis,
degenerative ataxias, cortical basal degeneration, ALS-Parkinson's-Dementia complex
of Guam, subacute sclerosing panencephalitis, Huntington's disease, Parkinson's disease,
synucleinopathies (including multiple system atrophy), primary progressive aphasia,
striatonigral degeneration, Machado-Joseph disease/spinocerebellar ataxia type 3 and
olivopontocerebellar degenerations, Gilles De La Tourette's disease, bulbar and pseudobulbar
palsy, spinal and spinobulbar muscular atrophy (Kennedy's disease), primary lateral
sclerosis, familial spastic paraplegia, Werdnig-Hoffmann disease, Kugelberg-Welander
disease, Tay-Sach's disease, Sandhoff disease, familial spastic disease, spastic paraparesis,
progressive multifocal leukoencephalopathy, familial dysautonomia (Riley-Day syndrome),
and prion diseases (including, but not limited to Creutzfeldt-Jakob, Gerstmann-Strussler-Scheinker
disease, Kuru and fatal familial insomnia) ,polyneuropathies (e.g. diabetic, alcohol-toxic,
Guillain-Barré-Syndrome, chronic inflammatory demyelinating polyneuropathy), prion
diseases, addiction, affective disorders (e.g. depression), schizophrenic disorders
, chronic fatique syndrome, chronic pain (e.g. lower back pain).
[0074] A further aspect of the present invention concerns the use of a polynucleotide, a
vector, a host cell or a polypeptide of the present invention for the manufacture
of an anti-aging medication. The basis for this application of the EPO variants of
the present invention is the fact that the progressing deterioration of most bodily
functions, which accompanies aging has been associated with cell death and it is,
therefore, envisioned that the EPO variants of the present invention, which only provide
the beneficial cell protective effect can be taken continuously without suffering
the side effects usually associated with the contnious administration of EPO, which,
however, can be attributed to the erythropoietic effect of EPO.
[0075] The inventors have astonishingly found that the nucleic acids and proteins according
to the invention possess astonishing anti-inflammatory properties (see figures and
experiments). Thus, these EPO variants are useful in treatment of inflammatory and
degenerative diseases. Inflammatory diseases are diseases such as but not limited
to multiple sclerosis, viral and bacterial infections or sepsis. Degenerative diseases
are diseases such as but not limited to stroke, myocardial infarctions.
[0076] The invention also relates to all kinds of forms of in vivo expression of the nucleic
acids according to the invention. It further relates to transformed cells, in particular
stem cells which are used as therapeutic agents. Such cells may be stably transformed
with a nucleic acid according to the invention. The nucleic acid may in a cassette
where it is operably linked to a promoter. The promoter may capable of driving the
expression only in particular tissues, such as but not limited to neuronal tissue
or the brain or tissue which exhibits inflammation or degeneration. Respective teaching
may be taken from
WO 97/14307.
[0077] The activity (in units) of EPO polypeptide is traditionally defined based on its
effectiveness in stimulating red cell production in rodent models (and as derived
by international standards of EPO). One unit (U) of regular EPO (MW of about.34,000)
is about10 ng of protein (1 mg protein is approximately 100,000 U). However, as mentioned
the invention involves the use of non-hematopoietic forms of erythropoietin, and as
such, this definition based on hematopoietic activity is inappropriate. Thus, as used
herein, the activity unit of EPO variant is defined as the amount of protein required
to elicit the same activity in neural or other erythropoietin-responsive cellular
systems as is elicited by native EPO in the same system. The skilled artisan will
readily determine the units of a non-hematopoietic EPO following the guidance herein.
[0078] In a further aspect the present invention provides a pharmaceutical composition comprising
a polynucleotide, a vector, a host cell, a polypeptide of the present invention and
a one or more pharmaceutically acceptable carrier.
[0079] In the practice of one aspect of the present invention, a pharmaceutical composition
as described above may be administered to a mammal by any route which provides a sufficient
level of an erythropoietin variant. It can be administered systemically or locally.
Such administration may be parenterally, transmucosally, e.g., orally, nasally, rectally,
intravaginally, sub-lingually, submucosally or transdermally. Preferably, administration
is parenteral, e.g., via intravenous or intraperitoneal injection, and also including,
but is not limited to, intra-arterial, intramuscular, intradermal and subcutaneous
administration. If the pharmaceutical composition of the present invention is administered
locally it can be injected directly into the organ or tissue to be treated. In cases
of treating the nervous system this administration route includes, but is not limited
to, the intracerebral, intraventricular, intracerebroventricular, intrathecal, intracistemal,
intraspinal and/or peri-spinal routes of administration, which can employ intracranial
and intravertebral needles, and catheters with or without pump devices.
[0080] In a preferred embodiment of pharmaceutical composition comprises an EPO variant
polypeptide in a dosage unit form adapted for protection or enhancement of EPO-responsive
cells, tissues or organs which comprises, per dosage unit, an effective non-toxic
amount within the range from about 0.5 mg to 5 mg of EPO variants; 0.6 mg to 5 mg
of EPO variants; 0.7 mg to 5 mg of EPO variants; 0.8 mg to 5 mg of EPO variants; 0.9
mg to 5 mg of EPO variants; 1 to 5 mg of EPO variants; 1.5 to 5 mg of EPO variants;
2 to 5 mg of EPO variants; 2.5 to 5 mg of EPO variants; 3.5 to 5 mg of EPO variants;
4 mg to 5 mg of EPO variants; or 4.5 to 5 mg of EPO variants and a pharmaceutically
acceptable carrier.
[0081] In a preferred embodiment, an EPO variant polypeptide may be administered systemically
at a dosage between 100 nanograms to about 50 micrograms per kg body weight, preferably
about 20 micrograms to about 50 micrograms per kg-body weight. Such serum levels may
be achieved at about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours post-administration. Such
dosages may be repeated as necessary. For example, administration may be repeated
daily, or every other, third, fourth, fifth, sixth, or seventh day, as long as clinically
necessary, or after an appropriate interval, e.g., every 1 to 12 weeks, preferably,
every 3 to 8 weeks. In one embodiment, the effective amount of EPO variant and a pharmaceutically
acceptable carrier may be packaged in a single dose vial or other container. Depending
on the respectively treated disease or condition the EPO variant can be administered
in a single dose, for a predetermined period of time or continuously. When an acute
condition or disease is treated it might be sufficient to provide the patient with
a single dose of EPO variant or for a period of, e.g. for 2 days to 12 months, preferably
1 week to 6 months, more preferably 2 weeks to 3 months. If a chronic disease or condition
is treated or if the EPO variant is used to prevent or reduce the deterioration associated
with aging the EPO variant can be administered continuously. If the EPO variant of
the present invention is administered for a given time period or continuously it is
preferably administered in the intervals and preferred intervals indicated above.
The intervals necessary will depend in part on the serum level of the EPO variant
necessary to treat or ameliorate the respective disease and on the pharmacokinetic
of the respective EPO variant, which will in part depend on modifications of EPO by,
for example, PEG. It will be in the discretion of the practitioner to determine the
exact duration, dose and type of EPO variant taking into consideration, e.g. the condition
of the patient to be treated, the severity of the dondition etc.
[0082] For other routes of administration, such as by use of a perfusate, injection into
an organ, or other local administration, a pharmaceutical composition will be provided
which results in similar levels of an EPO variant as described above. A level of about
10 pg/ml to about 1000 ng/ml is desired.
[0083] The pharmaceutical compositions of the invention may comprise a therapeutically effective
amount of a compound, e.g. polynucleotide, polypeptide, cell or vector, and a pharmaceutically
acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable"
means approved by a regulatory agency of the Federal or a state government or listed
in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals,
and more particularly in humans. The term "carrier" refers to a diluent, adjuvant,
excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical
carriers can be sterile liquids, such as saline solutions in water and oils, including
those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean
oil, mineral oil, sesame oil and the like. A saline solution is a preferred carrier
when the pharmaceutical composition is administered intravenously. Saline solutions
and aqueous dextrose and glycerol solutions can also be employed as liquid carriers,
particularly for injectable solutions. Suitable pharmaceutical excipients include
starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel,
sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,
propylene, glycol, water, ethanol and the like. The composition, if desired, can also
contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These
compositions can take the form of solutions, suspensions, emulsion, tablets, pills,
capsules, powders, sustained-release formulations and the like. The composition can
be formulated as a suppository, with traditional binders and carriers such as triglycerides.
The compounds of the invention can be formulated as neutral or salt forms. Pharmaceutically
acceptable salts include those formed with free amino groups such as those derived
from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed
with free carboxyl groups such as those derived from sodium, potassium, ammonium,
calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc. Examples of suitable pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically
effective amount of the compound, preferably in purified form, together with a suitable
amount of carrier so as to provide the form for proper administration to the patient.
The formulation should suit the mode of administration.
[0084] Pharmaceutical compositions adapted for oral administration may be provided as capsules
or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous
or nonaqueous liquids); as edible foams or whips; or as emulsions. Tablets or hard
gelatine capsules may comprise lactose, starch or derivatives thereof, magnesium stearate,
sodium saccharine, cellulose, magnesium carbonate, stearic acid or salts thereof.
Soft gelatine capsules may comprise vegetable oils, waxes, fats, semi-solid, or liquid
polyols etc. Solutions and syrups may comprise water, polyols and sugars.
[0085] An active agent intended for oral administration may be coated with or admixed with
a material that delays disintegration and/or absorption of the active agent in the
gastrointestinal tract (e.g., glyceryl monostearate or glyceryl distearate may be
used). Thus, the sustained release of an active agent may be achieved over many hours
and, if necessary, the active agent can be protected from being degraded within the
stomach. Pharmaceutical compositions for oral administration may be formulated to
facilitate release of an active agent at a particular gastrointestinal location due
to specific pH or enzymatic conditions.
[0086] Pharmaceutical compositions adapted for transdermal administration may be provided
as discrete patches intended to remain in intimate contact with the epidermis of the
recipient for a prolonged period of time. Pharmaceutical compositions adapted for
topical administration may be provided as ointments, creams, suspensions, lotions,
powders, solutions, pastes, gels, sprays, aerosols or oils. For topical administration
to the skin, mouth, eye or other external tissues a topical ointment or cream is preferably
used. When formulated in an ointment, the active ingredient may be employed with either
a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient
may be formulated in a cream with an oil-in-water base or a water-in-oil base. Pharmaceutical
compositions adapted for topical administration to the eye include eye drops. In these
compositions, the active ingredient can be dissolved or suspended in a suitable carrier,
e.g., in an aqueous solvent. Pharmaceutical compositions adapted for topical administration
in the mouth include lozenges, pastilles and mouthwashes.
[0087] Pharmaceutical compositions adapted for nasal administration may comprise solid carriers
such as powders (preferably having a particle size in the range of 20 to 500 microns).
Powders can be administered in the manner in which snuff is taken, i.e., by rapid
inhalation through the nose from a container of powder held close to the nose. Alternatively,
compositions adopted for nasal administration may comprise liquid carriers, e.g.,
nasal sprays or nasal drops. These compositions may comprise aqueous or oil solutions
of the active ingredient. Compositions for administration by inhalation may be supplied
in specially adapted devices including, but not limited to, pressurized aerosols,
nebulizers or insufflators, which can be constructed so as to provide predetermined
dosages of the active ingredient. In a preferred embodiment, pharmaceutical compositions
of the invention are administered via the nasal cavity to the lungs.
[0088] Pharmaceutical compositions adapted for rectal administration may be provided as
suppositories or enemas. Pharmaceutical compositions adapted for vaginal administration
may be provided as pessaries, tampons, creams, gels, pastes, foams or spray formulations.
Pharmaceutical compositions adapted for parenteral administration include aqueous
and nonaqueous sterile injectable solutions or suspensions, which may contain antioxidants,
buffers, bacteriostats and solutes that render the compositions substantially isotonic
with the blood of an intended recipient. Other components that may be present in such
compositions include water, alcohols, polyols, glycerine and vegetable oils, for example.
Compositions adapted for parenteral administration may be presented in unit-dose or
multi-dose containers, for example sealed ampules and vials, and may be stored in
a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid
carrier, e.g., sterile saline solution for injections, immediately prior to use. Extemporaneous
injection solutions and suspensions may be prepared from sterile powders, granules
and tablets. In one embodiment, an autoinjector comprising an injectable solution
of an EPO variant may be provided for emergency use by ambulances, emergency rooms,
and battlefield situations, and even for self-administration in a domestic setting,
particularly where the possibility of traumatic amputation may occur, such as by imprudent
use of a lawn mower. The likelihood that cells and tissues in a severed foot or toe
will survive after reattachment may be increased by administering an EPO variant to
multiple sites in the severed part as soon as practicable, even before the arrival
of medical personnel on site, or arrival of the afflicted individual with severed
toe in tow at the emergency room.
[0089] In a preferred embodiment, the composition is formulated in accordance with routine
procedures as a pharmaceutical composition adapted for intravenous administration
to human beings. Typically, compositions for intravenous administration are solutions
in sterile isotonic aqueous buffer. Where necessary, the composition may also include
a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the
site of the injection. Generally, the ingredients are supplied either separately or
mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free
concentrate in a hermetically-sealed container such as an ampule or sachette indicating
the quantity of active agent. Where the composition is to be administered by infusion,
it can be dispensed with an infusion bottle containing sterile pharmaceutical grade
water or saline. Where the composition is administered by injection, an ampule of
sterile saline can be provided so that the ingredients may be mixed prior to administration.
[0090] Suppositories generally contain active ingredient in the range of 0.5% to 10% by
weight; oral formulations preferably contain 10% to 95% active ingredient.
[0091] A perfusate composition may be provided for use in transplanted organ baths, for
in situ perfusion, or for administration to the vasculature of an organ donor prior
to organ harvesting
[0092] Such pharmaceutical compositions may comprise levels of an EPO variant or a form
of an EPO variant not suitable for acute or chronic, local or system administration
to an individual, but will serve the functions intended herein in a cadaver, organ
bath, organ perfusate, or in situ perfusate prior to removing or reducing the levels
of the EPO variant contained therein before exposing or returning the treated organ
or tissue to regular circulation.
[0093] The invention also provides a pharmaceutical pack or kit comprising one or more containers
filled with one or more of the ingredients of the pharmaceutical compositions of the
invention. Optionally associated with such container(s) can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals
or biological products, which notice reflects approval by the agency of manufacture,
use or sale for human administration.
[0094] In another embodiment, for example, EPO variant can be delivered in a controlled-release
system. For example, the polypeptide may be administered using intravenous infusion,
an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.
In one embodiment, a pump may be used (see
Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14: 201;
Buchwald et al. (1980) Surgery 88:507;
Saudek et al. (1989) N. Eng. J. Med. 321: 574). In another embodiment, the compound can be delivered in a vesicle, in particular
a liposome (see
Langer (1990) Science 249:1527-1533;
Treat et al. (1989) in Liposomes in the Therapy of Infectious Disease and Cancer,
Lopez-Berestein and Fidler (eds.), Liss, N.Y., 353-365;
WO 91/04014;
U.S. 4,704,355). In another embodiment, polymeric materials can be used (see
Medical Applications of Controlled Release (1974) Langer and Wise (eds.), CRC Press:
Boca Raton, Fla.;
Controlled Drug Bioavailability, Drug Product Design and Performance, (1984) Smolen
and Ball (eds.), Wiley: N.Y.;
Ranger and Peppas (1953) J. Macromol. Sci. Rev. Macromol. Chem. 23: 61; see also
Levy et al. (1985) Science 228:190; During et al. (1989) Ann. Neurol. 25: 351;
Howard et al. (1989) J. Neurosurg. 71: 105).
[0096] In another embodiment, EPO variant, as properly formulated, can be administered by
nasal, oral, rectal, vaginal, or sublingual administration.
[0097] In a specific embodiment, it may be desirable to administer the pharmaceutical compositions
of the invention locally to the area in need of treatment; this may be achieved by,
for example, and not by way of limitation, local infusion during surgery, topical
application, e.g., in conjunction with a wound dressing after surgery, by injection,
by means of a catheter, by means of a suppository, or by means of an implant, said
implant being of a porous, non-porous, or gelatinous material, including membranes,
such as silastic membranes, or fibers.
[0098] Selection of the preferred effective dose will be determined by a skilled artisan
based upon considering several factors which will be known to one of ordinary skill
in the art. Such factors include the particular form of the pharmaceutic composition,
e.g. polypeptide or vector, and its pharmacokinetic parameters such as bioavailability,
metabolism, half-life, etc., which will have been established during the usual development
procedures typically employed in obtaining regulatory approval for a pharmaceutical
compound. Further factors in considering the dose include the condition or disease
to be treated or the benefit to be achieved in a normal individual, the body mass
of the patient, the route of administration, whether administration is acute or chronic,
concomitant medications, and other factors well known to affect the efficacy of administered
pharmaceutical agents. Thus the precise dosage should be decided according to the
judgment of the practitioner and each patient's circumstances, e.g., depending upon
the condition and the immune status of the individual patient, according to standard
clinical techniques.
[0099] In another aspect of the invention, a perfusate or perfusion solution is provided
for perfusion and storage of organs for transplant, the perfusion solution including
an amount of an pharmaceutic compositions effective to protect EPO variant-responsive
cells and associated cells, tissues or organs.
[0100] Transplant includes but is not limited to xenotransplantation, where a organ (including
cells, tissue or other bodily part) is harvested from one donor and transplanted into
a different recipient; and autotransplant, where the organ is taken from one part
of a body and replaced at another, including bench surgical procedures, in which an
organ may be removed, and while ex vivo, resected, repaired, or otherwise manipulated,
such as for tumor removal, and then returned to the original location. In one embodiment,
the perfusion solution is the University of Wisconsin (UW) solution (
U.S. 4,798,824) which contains from about 1 to about 25 U/ml erythropoietin, 5% hydroxyethyl starch
(having a molecular weight of from about 200,000 to about 300,000 and substantially
free of ethylene glycol, ethylene chlorohydrin, sodium chloride and acetone); 25 mM
KH
2PO
4; 3 mM glutathione; 5 mM adenosine; 10 mM glucose; 10 mM HEPES buffer; 5 mM magnesium
gluconate; 1.5 mM CaCl
2. 105 mM sodium gluconate; 200,000 units penicillin; 40 units insulin; 16 mg Dexamethasone;
12 mg Phenol Red; and has a pH of 7.4-7.5 and an osmolality of about 320 mOSm/l. The
solution is used to maintain cadaveric kidneys and pancreases prior to transplant.
Using the solution, preservation may be extended beyond the 30-hour limit recommended
for cadaveric kidney preservation. This particular perfusate is merely illustrative
of a number of such solutions that may be adapted for the present use by inclusion
of an effective amount of the pharmaceutical composition. In a further embodiment,
the perfusate solution contains the equivalent from about 5 to about 35 U/ml erythropoietin,
or from about 10 to about 30 U/ml erythropoietin.
[0101] While the preferred recipient of an EPO variant for the purposes herein throughout
is a human, the methods herein apply equally to other mammals, particularly domesticated
animals, livestock, companion and zoo animals. However, the invention is not so limiting
and the benefits may be applied to any mammal.
[0102] If a person is known to be at risk of developing a stroke a prophylactic administration
of the pharmaceutical composition of the present invention is possible. In these cases
the pharmaceutical compositions, in particular EPO variant polypeptide is preferably
administered in above outlined preferred and particular preferred doses on a daily
basis. Preferably, between 100 nanograms to about 50 micrograms per kg body weight,
preferably about 20 micrograms to about 50 micrograms per kg-body weight. This administration
can be continued until the risk of developing a stroke has lessened. In most instances,
however, the pharmaceutical composition will be administered once a stroke has been
diagnosed. In these cases it is preferred that a first dose of the pharmaceutical
composition is administered for the first time within 24 hours after the first symptoms
of a stroke are evident, preferably within 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hour
or less. Preferably the administration is then continued for preferably at least 7,
more preferably at least 14 and more preferably for at least 21 days. The doses are
administered preferably once a day and preferably in above indicated doses.
Brief Description of the Figures and Drawings
[0103]
- Fig. 1:
- Comparison of EPO PCR products: Panel A depicts the DNA products of various PCR reactions
performed with either pure plasmid comprising the different murine EPO variants or
cDNA from mouse brain of kidney, which are separated on a 1.2% agarose gel. From the
left to the right the lanes comprise: 1 kb molecular weight marker, the product of
pure mK3, pure mG3, pure mG5, pure m301, pure mS, pure mWT, brain cDNA, kidney cDNA.
Panel B depicts the DNA product of a PCR performed with cDNA from human brain. From
the left to the right the lanes comprise 1 kb molecular weight standard and the PCR
product of human brain cDNA.
- Fig. 2:
- Alignment of nucleotide sequences of the EPO variants identified in murine brain cDNA
and "wild type" murine EPO, i.e. the sequence of the previously described EPO.
- Fig. 3:
- Alignment of nucleotide sequences of the EPO variants identified in human brain cDNA
and "wild type" human EPO.
- Fig. 4:
- Alignment of the amino acid sequences of the EPO variants identified in mouse and
human with the respective "wild type" EPO.
- Fig. 5:
- Hematopoietic activity of murine and human EPO and the EPO variants of the present
invention. Panel A depicts the results of a colony forming assay for murine EPO and
EPO variants and Panel B depicts the of a colony forming assay for human EPO and EPO
variants.
- Fig. 6:
- Experimental setup for neuroprotection assays with rhEPO and EPO-isomers.
- Fig. 7:
- Panel A shows an experiment with 1 h 40 min and 1 h 50 min oxygen glucose depriva-tion
(OGD) length. At both time-points a protection rate of 40-50% for the neuroguardians,
but no protection with mEPO and rhEPO was observed. Panel B shows an experiment with
two different time-points (OGD length between two experiments varies according to
density of neurons). At 2 h 45min only weak protection is achieved with wtEPO (20-30%)
compared to neuroguardians (60-70%). Full protection capacity of rh EPO is only observed
at higher damage levels (3 h 15min).
- Fig 8:
- Panel A shows an experiment with 2 h 00 min, 2 h 15 min and 2 h 20 min OGD length
with a protein concentration equalling 100 U/l hEPO. At all three time-points a protection
rate of 40-50% for the human, but no protection with mEPO and rhEPO. Panel B shows
an experiment with two different time-points (OGD length between two experiments varies
according to density of neurons). At 2 h 45 min only weak protection is achieved with
wtEPO (20-30%) compared to neuroguardians (60-70%). Full protection capacity of EPO
is only observed at higher damage levels (3 h 15 min).
- Fig 9:
- Panel A shows a Western Blot from medium of HEK293-cells transfected with pcDNA3.1-V5/His-hEPO, pcDNA3.1-V5/His-hS3 or pcDNA3.1-V5/His-hS4, respectively. These media were used for experiments shown in Fig. 8. Concentration
of hEPO, quantified by the mouse-EPO-ELISA (R&D) was 2U/ml. rhEPO (=2.5 ng were loaded
on the gel), hEPO (=0.4 ng were loaded on the gel), hS3 and hS4: each 20 µl medium
(collected 2 days after transfection). Marker = 5 µl BenchMark™ His-tagged Protein
Standard (Invitrogen). Panel B shows a Western Blot of His-Tag-purified mouse wild
type EPO (mEPO), human hS3 and hS4 EPO variants. mEPO was quantified with the EPO-mouse-ELISA.
130 pg mEPO were loaded onto the gel. (primary antibody: rabbit anti-rhEPO-Antikörper;
Santa-Cruz).
- Fig. 10:
- Alignment of the amino acid sequences of the EPO variants created recombinantly (alpha-helix
mutants) and identified in vivo. Herein SEQ ID NO 50 is the human alpha helix wild
type sequence; SEQ ID NO 51 is hAmA (point mutation Alanin); SEQ ID NO 52 is hAmE
(point mutation glutamic acid); SEQ ID NO 53 is hA-10 (deletion mutant) and SEQ ID
NO 54 is hA-20 (deletion mutant).
- Fig. 11:
- hEPO and hS3 mediated cytoprotection in a model of ischemia consisting of serum deprivation
and hypoxia in H9c2 cardiac myoblasts. H9c2 cells were incubated in serum-deprived
DMEM-medium either in normoxic or hypoxic conditions for 24h. Apoptosis was assessed
24h later by LDH assay. Data were normalized by setting the delta LDH release of untreated
cells in normoxic and hypoxic conditions to 100%. A: Column diagram representing the average values of normalized LDH release. B: Data were presented as box plot diagram showing the median (line across the box),
the 25th percentile (lower hinge), the 75th percentile (upper hinge), the maximum
and the minimum value. Number of experiments n=7. P*<0,001 (ANOVA1).
- Fig. 12:
- Immunoprecipitation of EPO variants using an anti-mEPO antibody from R&D (goat, biotin-labelled);
A: Detection of a second EPO isoform (30kDa) in a kidney protein extract of CoCl2-treated
mice (12956). B: Blocking of the antibody-antigen interaction by DarbpoietinA.
- Fig. 13:
- Neuroprotection mediated by Erythropoietin Alpha-helix (hA; n=4). hA 100/Ul: 30pM;
hA 50 U/l: 15pM; hEPO: 30pM=100U/l; P*<0,05; ANOVA1 versus control
- Fig. 14:
- Neuroprotection mediated by several human EPO-isoforms (n=6) P*<0,05; ANOVA1 versus
control
- Fig. 15:
- Neuroprotection mediated by Erythropoietin Alpha-helix deletion variants (n=6) P*<0,05;
ANOVA1 versus control A: column diagram showing the average values of normalized LDH release. B: Box plot showing the medians and percentiles (25%, 75%) values of normalized LDH
release
- Fig.16.
- Effects of human EPO variants and full length human EPO on LPS-induced cytokine production
by human macrophages. Purified human monocytes were differentiated into macrophages
in the presence of rhu M-CSF (50 ng/ml) for 6 days. Macrophages (1x106/ml) were pre-incubated
with hS4, hS3 or hEPO (300 mU/ml each) for 3h and then stimulated with 10 ng/ml endotoxin
(LPS from E.coli 0127:B8) for 4h. Cytokine concentration in supernatants was determined
by ELISA (Cytometric Beads Array , Becton Dickinson, Heidelberg, Germany). Data are
shown as mean±SD. ** Results differed from Control (PBS) group (p<0.01; Mann-Whitney
U test; n=3-9 per group).
- Fig. 17:
- Alignment of nucleic acid sequences of EPO deletion variants.
- Fig. 18:
- DNA sequences of mutants and deletion variants created recombinantly as well as wild
type Helix A (hWT-EPO Helix A). Herein SEQ ID NO 55 is hA (Wild type Helix A), SEQ
ID NO 56 is hAmA (deletion mutatant with Alanin), SEQ ID NO 57 is hAmE (deletion mutant
with glutamic acid), SEQ ID NO 58 is hA-10 (deletion mutant Helix A minus 10 aa) and
SEQ ID NO 59 is hA-20 (deletion mutant Helix A minus 20 aa).
- Fig. 19:
- A preferred embodiment wherein the leader transport sequences are deleted is depicted.
A shows the hA DNA sequence without leader as SEQ ID NO 60. This is the mature exported
protein. It also shows the leader-sequence (SEQ ID NO 63). B shows hA amino acid without
leader as SEQ ID NO 61 and the amino acid sequence of the leader-sequence as SEQ ID
NO 62.
Examples
Synthesis of murine EPO cDNA
[0104] RNA was isolated from kidneys of wild type C57BL/6 or SV129S6 mice or from two different
mouse brains (1 hour after stroke)by trizol extraction. The RNA was precipitated with
chloroform and isopropanol and finally dissolved in DEPC-H
2O. DNA was digested to the RQ1 RNase-free DNase protocol from Promega. The reaction
was stopped by addition of 200µl phenol/chloroform/isopropyl alcohol (25/24/1) to
the reaction mix and centrifugation for 10 min at 10000 rpm and 10°C. The supernatant
was mixed with 200 µl chloroform/isopropyl alcohol (24/1) and centrifuged for 10min
at 10000 rpm and 10°C. 20 µl 8 M lithium chloride and 550µl absolute ethanol were
added to the supernatant. This mix was then incubated for 1 h at -70°C and subsequently
precipitated for 30 min by centrifugation at 11000 rpm and 0°C. The resulting pellet
was washed with 600µl 75% ethanol, centrifuged at 8000 rpm (4°C, 10min) and dried
at room temperature. The RNA was dissolved in 20µl DEPC-H20.
[0105] Moloney murine leukemia virus reverse transcriptase (MuLV, RNase H minus, purchased
from Promega) was employed in first strand cDNA synthesis in a 15 µl reaction volume
with DEPC-H
2O comprising 3 µg RNA and 3 µl random hexamer primer (10 µM). Reverse transcription
was carried out with 6 µl M-MuLV reaction buffer (5x), 2 µl dNTP (2,5 mM each), 1
µl RNase inhibitor (1U/µl), 1 µl M-MuLV reverse transcriptase and 5 µl DEPC-H
2O in a PCR machine running the following program: 5 min at 21 °C; 1 h at 37°C; 5 min
at 95°C.
[0106] The resulting cDNA pool was used to amplify the complete EPO cDNA by a Nested PCR
approach. The first step employed primers lying outside of the coding region of the
EPO gene (genepo_sense (SEQ ID NO 39) gaa ctt cca agg atg aag act tgc age and genepo_antisense;
(SEQ ID NO 40): gtg gca gca gca tgt cac ctg tc). The second step used primers designed
to amplify the gene from start to stop codon, with attached
BamHI cleaving sites for the subsequent cloning (epo_sense (SEQ ID NO 41 tat gga tcc
atg ggg gtg ccc gaa cgt ccc ac and epo_antisense (SEQ ID NO 42 tat gga tcc tca cct
gtc ccc tct cct gca gac). All primers were from MWG-Biotech AG. A nested PCR was performed
in a Hybaid PCR machine in two steps, first PCR (3 min at 95°C; 35 cycles: 30 sec
at 65°C, 1 min at 72°C,30 sec at 95°C; 10 min at 72°C; 4°C hold) and second PCR (3
min at 95°C; 5 cycles: 30 sec at 67°C, 1 min at 72°C, 30 sec at 95°C; 15 cycles: 30sec
at 70°C, 1 min at 72°C, 30 sec at 95°C; 10 min at 72°C; 4°C).
[0107] In both PCRs,
Pfu Turbo Hotstart DNA Polymerase (Stratagene) was used according to the manufacturer's
protocol. The PCR product of the first step was diluted 1:50 for the second PCR. A
second cDNA synthesis protocol was performed using the Access RT-PCR System (Invitrogen)
with the following parameters: 48°C 5 min; 94°C 2 min; 40 cycles: 94°C 30 sec, 65°C
1 min, 70°C 2 min; 70°C 7 min; 4°C. The second PCR was performed as described above.
[0108] The amplified full-length EPO cDNA and the EPO isomers were separated on a 1.2% TAE-agarose
gel. A picture of the various PCR products is shown in Fig. 1a. The fragments were
than purified using the Wizard SV-Gel Cleanup System (Promega) or the Gel Extraction
Kit (Qiagen, Hilden, Germany). As
Pfu Polymerase generates blunt end products, the cDNA was subcloned in the pCR-Blunt
II-TOPO Vector using chemically competent Top10 One Shot Cells from (both Invitrogen).
[0109] Plasmid-DNA was isolated out of single colonies by usage of the Qiagen QIA prep Kit.
Inserts were sequenced on an ALFexpress™ DNA Sequencer (Pharmacia Biotech) using the
Thermo Sequenase™ Primer Cycle Sequencing Kit (Amersham Biosciences). The primers
M13FWDCY (SEQ ID NO 43: gtc gtg act ggg aaa acc ctg gcg) and M13REVCY (SEQ ID NO 44
agc gga taa caa ttt cac aca gga) were labelled with Cy5. The parameters for sequencing
were: t=900 min; T=55°C; 800V; 55 mA and 30 W. The sequence analysis revealed the
existence of a novel variant of EPO lacking exon 4 and three internally deleted variants.
The nucleotide sequences are depicted in Fig. 2a and Fig. 2b and the encoded peptide
sequences are depicted in Fig. 4. The nucleotide and peptide sequence of the EPO variant
mS corresponds to SEQ ID NO 13 and SEQ ID NO 14, respectively. The nucleotide and
peptide sequence of the EPO variant mG3 corresponds to SEQ ID NO 15 and SEQ ID NO
16, respectively. The nucleotide and peptide sequence of the EPO variant mG5 corresponds
to SEQ ID NO 17 and SEQ ID NO 18, respectively. The nucleotide and peptide sequence
of the EPO variant m301 corresponds to SEQ ID NO 19 and SEQ ID NO 20, respectively.
The nucleotide and peptide sequence of the EPO variant mK3 corresponds to SEQ ID NO
21 and SEQ ID NO 22, respectively.
Synthesis of human EPO cDNA
[0110] Human adult kidney (male) and fetal brain (male) poly A+ RNA was purchased from Stratagene.
cDNA was generated from 250 ng kidney RNA or 200 ng brain RNA according to the Moloney
murine leukaemia virus reverse transcriptase (MuLV, RNase H minus) as described above.
The resulting cDNA pool was used to amplify the complete EPO cDNA using
Pfu Polymerase (Stratagene) with the following primers: Hepo_sense (SEQ ID NO 45): gat
ggg ggt gca cga atg tcc tgc and Hepo_antisense (SEQ ID NO 46): cac acc tgg tca tct
gtc ccc tgt c.
[0111] The PCR was performed in a PCR machine from Invitrogen (3 min at 95°C; 35 cycles:
30 sec at 67°C, 1 min at 72°C, 30 sec at 95°C; 10 min at 72°C). In the case of the
fetal brain cDNA a Nested PCR approach was used, performing a second amplifying step
on the PCR product of 20 cycles. The amplified PCR products were separated on a 1.2%
TAE-agarose gel (Fig. 1b) and purified using the Gel Extraction Kit (Qiagen, Hilden,
Germany). The purified cDNA was subcloned in the pCR-Blunt II-TOPO Vector using chemically
competent Top10 One Shot Cells (both from Invitrogen). Plasmid-DNA was isolated out
of single colonies by usage of the QIA prep Kit (Qiagen, Hilden, Germany). Inserts
were sequenced on an ALFexpress™ DNA sequencer (Pharmacia Biotech) using the Thermo
Sequenase ™ Primer Cycle Sequencing Kit (Amersham Biosciences). The primers M13FWDCY
(SEQ ID NO 43) and M13REVCY (SEQ ID NO 44) were labelled with Cy5. The parameters
for sequencing were: t=900min; T=55°C; 800 V; 55 mA and 30 W. The sequence analysis
revealed the existence of two novel variants of human EPO missing exon 3 and the first
half of exon 4, respectively, and a number of variants that follows the rule of repeated
trimers or hexamers as detected in the mouse. The nucleotide sequences are depicted
in Fig. 3a and Fig. 3b and the encoded peptide sequences are depicted in Fig. 4. The
nucleotide and peptide sequence of the EPO variant hS3 corresponds to SEQ ID NO 1
and SEQ ID NO 2, respectively. The nucleotide and peptide sequence of the EPO variant
h1-4 corresponds to SEQ ID NO 3 and SEQ ID NO 4, respectively. The nucleotide and
peptide sequence of the EPO variant h1-5 corresponds to SEQ ID NO 5 and SEQ ID NO
6, respectively. The nucleotide and peptide sequence of the EPO variant hS4 corresponds
to SEQ ID NO 7 and SEQ ID NO 8, respectively. The nucleotide and peptide sequence
of the EPO variant h1-1 corresponds to SEQ ID NO 9 and SEQ ID NO 10, respectively.
The nucleotide and peptide sequence of the EPO variant h2-1 corresponds to SEQ ID
NO 11 and SEQ ID NO 12, respectively.
Expression of His-tagged proteins in HEK cells
[0112] BamHI and EcoRI restriction sites for cloning were added to both the mouse and the
human EPO variants by using overhang sense primers and overhang antisense primers
without stop codon
(for mouse variants: epo_sense (SEQ ID NO 41) and epoeco_antisense (SEQ ID NO 47): aaa gaa ttc cct gtc
ccc tct cct gca gac ctc;
for human variants; hepobam_se (SEQ ID NO 48): tat gga tcc atg ggg gtg cac gaa tgt cc, hepoeco_as [SEQ
ID NO 49]: aga gaa ttc tct gtc ccc tgt cct gca g). The PCR products were cloned into
pcDNA-3.1-HIS/V5 A (Invitrogen) using BamHI and EcoRI restriction sites. Plasmids
were amplified in XL-1 Blue Competent Cells (recA1 endA1 gyrA96 thi1 hsdR17 supE44
relA1 lac [F' proAB lacl
qZΔM15 Tn10 (Tet
R)]) (Stratagene). The XL-1 Blue Competent Cells transformation protocol was performed
without β-mercaptoethanol and with a prolonged heat pulse of 60 seconds. Plasmid DNA
was extracted using the QIAprep Spin Miniprep Kit (Qiagen, Hilden, Germany). For transfection
into mammalian cells DNA was extracted using the EndoFree Plasmid Maxi Kit (Qiagen,
Hilden, Germany). HEK 293 cells (BD biosciences) were grown for 18 days in Dulbecco's
modified Eagle's medium (DMEM; Biochrom, Berlin, 1 g/l glucose, 3.7 g/l NaHCO
3; supplemented with 10% fetal calf serum GOLD, 1% penicillin/streptavidine and 1%
L-glutamine) in tissue culture flasks (25 cm
2) at 37°C and 5% CO
2. Cells were split every 2-3 days after reaching 80-90% confluence. At DIV18 120,000
cells were plated per well in a 12 well plate containing Dulbecco's modified Eagle's
medium without antibiotics. Cells were grown for approximately 48 h till 50% confluence.
Transfection was performed with Lipofectamine 2000 (Invitrogen) adapting the provided
protocol for HEK cells.
[0113] Plating medium of HEK-cells was replaced 10 min before transfection by serum-free
DMEM without antibiotics. Cells were incubated 5 h at 37°C with DNA-Lipofectamine
complexes. Medium was then changed to fresh serum-containing DMEM without antibiotics.
At DIV2 cells were split and plated in Dulbecco's modified Eagle's with antibiotics.
Expression and Purification of His-tagged EPO variants
[0114] His-tagged proteins were transiently expressed in HEK-cells. Medium from HEK293 cells
was harvested 2-6 days after transfection with pcDNA-3.1-HIS/V5 A - constructs. Cell
debris was pelleted at 3500 rpm, 4°C for 15 min. BD TALON™ Metal Affinity Resin (BD
Biosciences) was used for purification of his-tag proteins. All steps (equilibration,
washing and elution) were performed at pH 7.1. The provided protocol was modified
to a prolonged over-night binding step at 4°C. Eluate was collected in 500 µl-fractions.
Fractions were analysed by Western Blots using an anti-rhEPO antibody from Santa-Cruz
or a murine EPO ELISA-Kit (R&D). Imidazole was removed from protein-containing fractions
using HiTrap™ Desalting columns (5 ml) from Amersham Biosciences according to the
manufactures protocol. This included a change of buffer to PBS.
Western Blot
[0115] A 16% SDS-Gel was prepared using standard-protocols and run at 110 V. Blotting was
done on nitrocellulose-membranes for 45 min at 200 mA. The blot was blocked for at
least one hour in blocking buffer containing 5% non-fat dry milk powder in 0,1% Tween-20.
Incubation with the first antibody (EPO (H-162) sc-7956 rabbit polyclonal IgG, Santa
Cruz, 1:500) was performed over-night at 4°C. The secondary antibody (goat anti-rabbit
HRP; 1:1000) was added for 2 hours at room-temperature. The blot was revealed by use
of Luminol; photos were exposed for 2 minutes. Membranes were stained with Ponceau
Red. The EPO specific antibody was capable of detection all EPO variants.
Erythroid Colony formation assay
[0116] Bone marrow cells were harvested from tibia and femur of male C57BL/6 mice (8-11
weeks) and resuspended in α-medium (supplemented with 10% fetal calf serum GOLD, 1%
penicillium/streptavidine and 1% L-glutamine). Cells were seeded in 35 mm
2 Petri dishes (225.000 cells/dish) containing 8 parts Metho Cult SF 3236 methyl cellulose
(StemCell Technologie Inc), 1 part cells and 2 parts α-medium mixed with HEK-cell
preconditioned medium containing the EPO derivates (150 U/l in the case of murine
EPO). 150 U/l of rhEPO (Roche) was used as positive control. Plates were incubated
at 37°C in a humidified atmosphere containing 5% CO
2 for 48 hours. For evaluation only reddish colonies containing at least 6 hemoglobinised
cells were taken into account.
Hematopoietic potential of the EPO variants
[0117] Metho Cult SF 3236 triggers the formation of colonies (CFU-M, CFU-G or CFU-E) only
after addition of the appropriate cytokines. Formation of CFU-E (Colony forming unit-erythroblast)
can be observed, after addition of erythropoietin, after 2 days. The small irregular
reddish colonies disappear by day 3.
[0118] In this assay, the hematopoietic potential of the variants was tested and compared
to wild type form of EPO as well as rhEPO. The following conditions were prepared
for comparison: medium from HEK cells transfected with pZ/EG as negative control,
medium from HEK cells transfected with pZ/EG cells plus 150U/l rhEPO (Roche) as a
positive control, and medium from HEK cells transfected with either pZ/EG-EPO-IRES
(150U/l murine EPO), pZ/EG-Splice-IRES (variant S; mS) or pZ/EG-G3-IRES (variant G3;
mG3). At DIV2 only reddish colonies were counted containing at least 6 hemoglobinised
cells. The results of three independent experiments are depicted in Fig. 5.
[0119] In comparison to murine EPO and rhEPO the murine EPO variants (mS and mG3-variant)
lacked haematopoietic potential.
Primary neuronal cultures
[0120] Rat primary neuronal cultures were obtained from E16 to early E19 embryos of Wistar
rats (Bundesinstitut für gesundheitlichen Verbraucherschutz und Veterinärmedizin,
Berlin, Germany). Cre-expressing mouse neurons were obtained from E16 embryos of heterozygous
transgenic mice expressing Cre-recombinase under the control of the tubulin α-1 promoter
(provided by Dr. U. Schweitzer; Experimental Endocrinology, Charité). Murine and rat
cultures were prepared according to a modified protocol from
Brewer (1995) J Neurosci Res. 42: 674-83. Cerebral cortex was isolated after removal of meninges and rinsed twice in PBS (Biochrom,
Berlin, Germany). After 15 min incubation in trypsin/EDTA (0.05/0.02% w/v in PBS)
at 37°C, tissues were rinsed twice in N-Med (modified Eagle's medium from Gibco with
10% fetal calf serum, 100 U penicillin plus streptomycin from Biochrom, 2 mM L-glutamine,
100 IE insulin/l, 10 mM HEPES and 44 mM glucose) and dissociated carefully in a small
volume of N-Med using a Pasteur pipette. Cells were pelleted at room temperature by
2 min centrifugation at 210 g and resuspended in NBM starter medium (Neurobasal medium
from Gibco with 2% B27 supplement from Gibco, 1% Pen/Strep, 0,5 mM L-glutamine and
25 µM glutamate).
Preparation of culture plates
[0121] 24-well plates and 6-well plates were pretreated by over-night incubation at 4°C
with poly-L-lysin from Biochrom (2.5 µg/ml in PBS). Rinsing of the wells with PBS
was followed by 1h incubation at 37°C with coating medium (modified Eagle's medium
with 5% FCS Gold from PAA, 1% Pen/Strep, 10mM HEPES and 0,03 w/v collagen G from Biochrom),
then the wells were carefully rinced twice with PBS. Volume and type of plating medium
was chosen depending on experimental procedure.
Oxygen glucose deprivation in rat primary cortical neurons - a cell culture model
of cerebral ischemia
[0122] For OGD the culture medium was washed out by rinsing once with PBS. OGD was induced
with 500 µl of a deoxygenated aglycemic solution (BSS
0-O
2; 143.8 mM Na
+, 5.5 mM K
+, 1.8 mM Ca
2+, 0.8 mM Mg
2+, 125.3 mM Cl
-, 26.2 mM HCO
3- and 0.8 mM SO
42-, pH 7.4) in a hypoxic atmosphere generated by a dedicated, humidified gas-tight incubator
(Concept 400, Ruskinn Technologies, Bridgend, UK) flushed with a gas mix containing
5% CO
2, 85% N
2 and 10% H
2. OGD-time depended on the density and the age of the culture and varied between 2
h 30min and 2 h 40min. In control experiments the wells were treated with 500 µl of
the oxygenated glycemic BSS
0 solution (BSS
0+O
2; 143.8 mM Na
+, 5.5 mM K
+, 1.8 mM Ca
2+, 0.8 mM Mg
2+, 125.3 mM Cl
-, 26.2 mM HCO
3-,0.8 mM SO
42-, and 20 mM glucose, pH 7.4) and incubated at 37°C in a normoxic atmosphere containing
5% CO
2. Immediately after OGD, treated cells and controls were changed from BSS solution
to 500µl of medium containing 40% conditioned NBM plus 60% fresh NBM. After 24h, lactate
dehydrogenase (LDH) activity was measured in the supernatants as an indicator of cell
death.
[0123] For LDH measurement 25 µl of medium was mixed with 100 µl fresh β-NADH solution (0.15
mg/ml in 1xLDH-buffer; Sigma, reduced form) in a 96 wells plate (Greiner). 25 µl of
22.7 mM pyruvate (Sigma) was added immediately before placing the plate into the Reader
(Thermo Labsystems; MRX
TC Revelation). Parameters were chosen as follows: filter: 340 nm, shake time: 5 sec,
interval: 30 sec, counts: 10. LDH-concentration was calculated proportionally to the
LDH-standard (Greiner, system calibrator).
Induction of neuroprotection by conditioned medium from transfected HEK293 cells expressing
EPO variants
[0124] In the following experiments rhEPO (recombinant human EPO from Sigma Aldrich, Deisenhofen,
Germany) was used as a positive control. Neuroprotection assays are schematically
depicted in Fig. 6. Neurons were plated in 24-well plates at a density of 300,000
cells in a final volume of 600 µl NBM starter medium. After 4 days, 200 µl of the
medium was replaced by 250 µl fresh NBM (same as NBM starter without glutamate).
[0125] For pretreatment with rhEPO, wild type mEPO, wild type hEPO or EPO variants the medium
was removed to an end volume of 200 µl and filled up with 200 µl fresh NBM+B27 containing
equimolar amounts (corresponding to 200 U/l rhEpo) of EPO or EPO variants, respectively.
Equivalent concentrations of the various EPO variants (as well as mEPO and hEPO) in
the conditioned medium from HEK293 cells were estimated by Western blot and EPO-Elisa.
Thereafter neurons were grown for 48 h under normoxic, humified conditions at 37°C
before oxygen glucose deprivation (OGD) was performed (OGD interval as indicated).
Cell death was assessed 24 h after OGD by measurement of LDH release. Reduction in
LDH release, compared to mock-treated neurons (medium from HEK293 cells transfected
with the backbone plasmid; = ko; 100%), is a quantitative measure of neuroprotection.
In all experiments we observed a more robust neuroprotective effect provided by the
EPO variants, if compared to wt EPO (see Fig. 7 Panel A and B for murine EPO and variants
thereof and Fig. 8 Panel A and B for human EPO and variants thereof).
[0126] The neuroprotection induced by the murine EPO variants is more robust than that induced
by EPO (rhEPO as well as wild type mouse EPO). For example, neuroprotection mediated
by EPO can only be observed in a clearly defined window of OGD length (corresponding
to a clearly defined damage level). At low concentration the neuroprotection by hS3
and hS4 was equal or better than the neuroprotection of wt hEPO. Overall, neuroprotection
induced by the variants is stronger than that induced by rhEPO. In addition, variants
have an higher neuroprotective potential than both wild type forms mEPO and hEPO,
which were produced by the same procedure as the EPO variants.
H9c2 - model of ischemia
[0127] The rat BDIX heart myoblast cell line (obtained from European Collection of Cell
Cultures) was cultured in DMEM (Biochrom) containing 4.5g/l glucose supplemented with
2mM L-glutamine, 10% inactivated fetal calf serum and 1% penicillin-streptavidin.
Subconfluent cultures (70%) were subcultured 1:4. Cells were plated in 400µl medium
containing 120pM hEPO or hS3 respectively in a density of 15,000 cells per well in
24-well plates and cultured for 48 hours. Hypoxia was achieved by culturing the cells
in 400µl serum-deficient DMEM containing 4.5g/l glucose supplemented with 2mM L-glutamine
and 1% penicillin-streptavidin and leaving them for 24h in an anaerobic workstation
(Concept 400, Ruskinn Technologies, Bridgend, UK) saturated with a gas mix containing
5% CO
2, 85% N
2 and 10% H
2 at 37°C. Control cells were left in serum-deficient DMEM in a normoxic incubator.
At the end of the experiment medium was replaced to 400µl fresh serum-deficient DMEM
and LDH was measured according to standard protocols 24h later.
Immunoprecipitation
[0128] Male 129S6 mice or male C57BI6 mice (8-10 weeks, Bundesinstituts für Risikobewertung,
Berlin) having free access to food and water were used for the experiments. CoCl2
was injected subcutanely in a dose of 60mg/kg and animals were killed 18 hours later.
Protein expression was measured in serum, kidney and brain protein extracts by a commercial
available ELISA (R&D, mEPO).
[0129] Antibodies for immunoprecipitation were purchased from R&D (anti-mEPO antibody, goat,
biotin-labelled) and Santa-Cruz (anti-rhEPO, rabbit). Immunoprecipitation was perfomed
according to standard protocols and evaluated by western blot.
[0130] Blocking of the western blot detection antibody was achieved by two hours incubation
with 10µg DarbpoietinA at room temperature prior to blot incubation.
Generation of alpha-helix-mutants (Fig. 10)
[0131] Human alpha-helix-mutants were all generated by PCR based approaches using standard
protocols.
[0132] Mutant A (hAmA) and mutant E (hAmE) are variants of the alpha-helix with amino acid
exchange at position 41 (arginine). cDNA sequence was changed from AGG to GCG for
mutant A (alanine) or to GAG for mutant E (glutamate). -20aa and -10aa are deletion
variants of the alpha-helix missing 20 amino acids or 10 amino acids respectively
at the c-terminus. All mutants were generated without V5 and His-tag and expressed
in HEK 293 cells. Neuroprotection experiments were performed as described previously
using medium of transfected HEK cells expressing the different variants.
hEPO and hS3 mediated cytoprotection in a model of ischemia in H9c2 cells (Fig. 11)
[0133] The cytoprotective potential of the EPO variants was shown exemplarily for purified
hEPO and hS3 in a model of ischemia consisting of serum deprivation and hypoxia in
H9c2 cardiac myoblasts (Figure 1). LDH release was assessed as a marker of apoptotic
cell death. We found significant cytoprotective capacities for both variants (approximately
20% and 25% for hEPO and hS3).
Immunoprecipitation reveals EPO splicing isoform in kidney protein extracts of CoCl2-treated
mice (Fig. 12)
[0134] To strengthen our finding of EPO splicing isoforms in human and murine tissues by
a PCR-based approach we performed immunoprecipitations on murine serum, brain and
kidney protein extracts of CoCl2 treated mice using antibodies tested to recognize
both isoforms. Subcutane injection of CoCl2 is known to increase erythropoietin levels
in several mouse tissues, namely blood, brain, liver and kidney.
[0135] We were able to precipitate erythropoietin (approximately 40kDa) from serum, brain
and kidney protein extracts of CoCl2 treated mice (Figure2); precipitation of erythropoietin
from a kidney protein extract of an untreated mouse failed due to the low expression
level. Furthermore we were able to prove the existence of a second smaller protein
(approximately 30kDa) in the kidney protein extract of CoCl2 treated mice. This protein
is specifically recognized by the anti-rhEPO antibody as shown by complete blocking
of the antibody-antigene interaction with Darbpoietin A. These findings strongly support
the existence of a murine erythropoietin splicing isoform. These results were reproduced
in a second mouse strain, namely C57BI6.
Neuroprotection mediated by different isoforms of the erythropoietin alpha-helix (Fig.
13)
[0136] Analysing the neuroprotective potentials of the so far identified erythropoietin
variants we suggested the alpha helix to be the functionally important domain for
the neuroprotective character of erythropoietin. In order to test this hypothesis
we expressed a shortened form of human erythropoietin, namely the alpha-helix domain,
in HEK 293 cells and tested this peptide in our OGD-model. We found an equivalent
protective potential with 30pM and 15pM of this peptide to 30pM of hEPO as shown in
Figure 13.
[0137] In order to identify the functional important residues in the alpha helix domains
of human erythropoietin we generated different erythropoietin mutants containing either
amino acid exchanges (hAmA and hAmE) or complete domain deletions (hA-10 and hA-20).
[0138] Neither the neutral nor the acidic amino acid exchange at position 41 was able to
destroy the neuroprotective potential of the alpha-helix in our OGD model (Figure
14).
Neuroprotection mediated by several human EPO-isoforms (n=6) P*<0,05; ANOVA1 versus
control (Fig. 14)
[0139] Deletion variants missing 10 or 20 amino acids at the c-terminus of the alpha-helix
were expressed in HEK293 cells and also tested in the OGD-model. The deletion variant
hA-10 had still neuroprotective properties comparable to the hS3 splice isoform. Deletion
of 20 amino acids (hA-20) led to a peptide that was not protective anymore (Fig. 15).
Immunomodulation by human erythropoietin variants (Fig. 16)
[0140] The human EPO variants hS3 and hS4 exhibit strong immunomodulatory effects. In human
macrophages stimulated with the endotoxin lipopolysaccharide (LPS) hS3 and hS4 induce
the anti-inflammatory cytokine IL-10 and reduce the expression of the pro-inflammatory
cytokines IL-6 and IL-8. Compared to EPO (hWT) anti-inflammatory effects of hS3 and
hS4 are much more pronounced. These anti-inflammatory properties of EPO variants are
useful in treatment of inflammatory (e.g. Multiple Sclerosis, viral and bacterial
infections, sepsis) and degenerative diseases (e.g. stroke, myocardial infarctions).
SEQUENCE LISTING
[0141]
<110> Charité - Universitatsmedizin Berlin
<120> Erythropoietin Splice Variants
<130> U60011PCT
<160> 63
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Gly Asp Arg
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1. Ein Polynukleotid, das eine Erythropoietin (EPO)-Variante kodiert, aus der Gruppe
ausgewählt bestehend aus:
(a) Polynukleotiden, die die reife Form der Polypeptide kodieren, welche als hs3,
h1-4, h1-5, hs4, h1-1, h2-1, mS, mG3, mG5, m301, mK3, ha, hAma, hAme und hA-10 bezeichnet
werden und die abgeleitete Aminosäuresequenz wie in SEQ ID Nr: 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 22, 50, 51, 52, bzw. 53 gezeigt besitzen;
(b) einem Polynukleotid, das die reife Form des Polypeptids kodiert, welches als ha-Sequenz
ohne Leader bezeichnet ist und aus der abgeleiteten Aminosäuresequenz wie in SEQ ID
Nr: 61 gezeigt besteht;
(c) Polynukleotiden mit der kodierenden Sequenz wie in SEQ ID Nr: 1,3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 55, 56, 57 und 58 gezeigt, die mindestens die reife Form des Polypeptids
kodieren;
(d) einem Polynukleotid, bestehend aus der kodierenden Sequenz wie in SEQ ID Nr: 60
gezeigt, welches die reife Form des als ha-Sequenz ohne Leader bezeichneten Polypeptids
kodiert;
(e) Polynukleotid, welches eine humanisierte Version der Polypeptide mS, mG3, mG5,
m301 und mK3, bestehend aus der abgeleiteten Aminosäuresequenz wie in SEQ ID Nr: 14,
16, 18, 20 und 22 gezeigt, kodiert;
(f) Polynukleotiden, die ein Polypeptid kodieren, welches eine Fusion einer Aminosäuresequenz,
ausgewählt aus der Gruppe von Aminosäuresequenzen wie in SEQ ID Nr: 24, 26, 28 und
30 gezeigt, an den N-Terminus einer Aminosäuresequenz, ausgewählt aus der Gruppe von
Aminosäuresequenzen wie in SEQ ID NO: 32, 34, 36 und 38 gezeigt, umfassen, wobei die
Fusion zellschützende und insbesondere neuroprotektive Aktivität, aber im Wesentlichen
keine hämatopoetische Aktivität hat;
(g) Polynukleotiden, die eine Fusion von Polynukleotidsequenzen ausgewählt aus der
Gruppe von Polynukleotidsequenzen wie in SEQ ID Nr: 23, 25, 27 und 29 gezeigt an das
5'-Ende einer Polynukleotidsequenz ausgewählt aus der Gruppe von Polynukleotidsequenzen
wie in SEQ ID NO: 31, 33, 35 und 37 gezeigt, umfassen, wobei die Fusion zellschützende
und insbesondere neuroprotektive Aktivität, aber im Wesentlichen keine hämatopoetische
Aktivität hat;
(h) Polynukleotiden, die ein Derivat eines Polypeptids kodieren, welches durch ein
Polynukleotid nach einem der Punkte (a) bis (g) kodiert ist, wobei in dem Derivat
zwischen 1 und 10 Aminosäurereste, im Vergleich zu dem Polypeptid, konservativ substituiert
sind und das Derivat zellschützende und insbesondere neuroprotektive Aktivität, aber
im Wesentlichen keine hämatopoetische Aktivität hat;
(i) Polynukleotiden, die ein Fragment eines Polypeptids kodieren, welches durch ein
Polynukleotid nach einem der Punkte (a) bis (h) kodiert ist, wobei in dem Fragment
zwischen 1 und 10 Aminosäurereste N- und/oder C-terminal deletiert sind und/oder zwischen
1 und 10 Aminosäuren N- und/oder C-terminal von der Übergangsstelle, verglichen mit
dem Polypeptid, deletiert sind und das Fragment zellschützende und insbesondere neuroprotektive
Aktivität, aber im Wesentlichen keine hämatopoetische Aktivität hat;
(j) Polynukleotiden, die zu mindestens 95% identisch sind mit einem Polynukleotid
nach einem der Punkte (a) bis (d) und die zugleich zellschützende und insbesondere
neuroprotektive Aktivität, aber im Wesentlichen keine hämatopoetische Aktivität haben;
(k) Polynukleotiden, deren komplementärer Strang unter stringenten Bedingungen mit
einem Polynukleotid wie in einem der Punkte (a) bis (j) definiert hybridisiert und
die für ein Polypeptid kodieren, welches zellschützende und insbesondere neuroprotektive
Aktivität, aber im Wesentlichen keine hämatopoetische Aktivität hat;
(l) Polynukleotiden, die ein EPO-Variante-Polypeptid kodieren, welches den N-terminalen
Teil von Volllängen-EPO einschließlich Helix A umfasst und dem mindestens eines der
folgenden fehlt:
(i) ein Fragment von mindestens 10 Aminosäuren zwischen Helix A und B-Helix;
(ii) ein Fragment von mindestens 10 Aminosäuren von Helix B;
(iii) ein Fragment von mindestens 6 Aminosäuren zwischen Helix B und Helix C;
(iv) ein Fragment von mindestens 10 Aminosäuren von Helix C;
(v) ein Fragment von mindestens 20 Aminosäuren zwischen Helix C und D,
und/oder
(vi) ein Fragment von mindestens 10 Aminosäuren von Helix D;
wobei die Variante zellschützende und insbesondere neuroprotektive Aktivität, aber
im Wesentlichen keine hämatopoetische Aktivität hat;
(m) Polynukleotiden, die ein Derivat eines Polypeptids kodieren, welches durch ein
Polynukleotid nach (1) kodiert ist, wobei in dem Derivat zwischen 1 und 10 Aminosäurereste
konservativ substituiert sind, verglichen mit dem Polypeptid, und das Derivat zellschützende
und insbesondere neuroprotektive Aktivität, aber im Wesentlichen keine hämatopoetische
Aktivität hat;
(n) Polynukleotide, deren komplementärer Strang unter stringenten Bedingungen mit
einem Polynukleotid wie in einem von (1) bis (m) definiert hybridisieren und die für
ein Polypeptid kodieren, welches zellschützende und insbesondere neuroprotektive Aktivität,
aber im Wesentlichen keine hämatopoetische Aktivität hat;
oder der komplementäre Strang eines solchen Polynukleotids.
2. Das Polynukleotid nach Anspruch 1, welches DNA, genomische DNA oder RNA ist.
3. Ein Vektor, der das Polynukleotid nach Anspruch 1 oder 2 enthält.
4. Der Vektor nach Anspruch 3, in dem das Polynukleotid 1 operativ mit Expressionskontrollsequenzen
verbunden ist, die die Expression in prokaryontischen und/oder eukaryontischen Wirtszellen
ermöglichen.
5. Eine Wirtszelle, die mit dem Polynukleotid nach Anspruch 1 oder 2 oder dem Vektor
nach Anspruch 3 oder 4 genetisch verändert worden ist.
6. Ein transgenes nicht-menschliches Säugetier, das aus der Gruppe aus nichtmenschlichen
Primaten, Pferd, Rind, Schaf, Ziege, Schwein, Hund, Katze, Kaninchen, Maus, Ratte,
Meerschweinchen, Hamster und Rennmaus ausgewählt wird, welches das Polynukleotid nach
Anspruch 1 oder 2, einen Vektor nach Anspruch 3 oder 4 und/oder einer Wirtszelle nach
Anspruch 5 umfasst.
7. Ein Verfahren zur Herstellung eines EPO-Varianten-Polypeptids, welches durch das Polynukleotid
nach Anspruch 1 oder 2 kodiert wird, umfassend: Kultivieren der Wirtszelle nach Anspruch
5 und Gewinnen des durch das Polynukleotid kodierten Polypeptids.
8. Das Verfahren nach Anspruch 7, das ferner den Schritt umfasst, die EPO-Variante zu
modifizieren, wobei die Modifikation aus der Gruppe ausgewählt wird, die aus Oxidation,
Sulfatierung, Phosphorylierung, Addition von Oligosacchariden oder Kombinationen davon
besteht.
9. Verfahren zur Herstellung von Zellen, die zur Expression mindestens einer der EPO-Varianten
fähig sind, welches eine genetische Veränderung von Zellen in vitro mit dem Vektor nach Anspruch 3 oder 4 umfasst, wobei das/die EPO-Varianten-Polypeptide(e)
durch das Polynukleotid nach Anspruch 1 oder 2 kodiert ist (sind).
10. Ein Polypeptid mit einer Aminosäuresequenz, die durch das Polynukleotid nach Anspruch
1 oder 2 kodiert wird oder durch das Verfahren nach Anspruch 7 oder 8 erhältlich ist.
11. Pharmazeutische Zusammensetzung, umfassend das Polynukleotid nach Anspruch 1 oder
2, einen Vektor nach Anspruch 3 oder 4, eine Wirtszelle nach Anspruch 5, und/oder
ein Polypeptid nach Anspruch 10 und einen oder mehreren pharmazeutisch verträgliche
Trägerstoff(e).
12. Verwendung des Polynukleotids nach Anspruch 1 oder 2, eines Vektors nach Anspruch
3 oder 4, einer Wirtszelle nach Anspruch 5 oder eines Polypeptids nach Anspruch 10
zur Herstellung eines Medikaments zur Behandlung oder Prävention eines Zustands, der
mit Gewebeschädigung infolge von Zelltod assoziiert ist.
13. Verwendung nach Anspruch 12, wobei der Zelltod induziert wird durch Ischämie, Hypoxie,
bakterielle Infektionen, virale Infektionen, autoimmunologisch, traumatisch, chemisch
induziert oder strahlungsinduziert.
14. Verwendung nach den Ansprüchen 12 oder 13, wobei der Zustand eine akute oder chronische
neurodegenerative und/oder neuroinflammatorische Störung ist, eine akute oder chronische
Erkrankung des Herzens, der Lunge, der Niere, der Leber oder der Bauchspeicheldrüse
ist oder der Zustand mit einer Organ- oder Zelltransplantation assoziiert ist.
15. Verwendung nach Anspruch 14, wobei die akute neurodegenerative und/oder neuroinflammatorische
Störung aus der Gruppe ausgewählt wird, die aus zerebraler Ischämie oder Infarkt,
einschließlich embolischen Verschlusses und thrombotischen Verschlusses, Reperfusion
nach einer akuten Ischämie, perinatale hypoxisch-ischämische Verletzung, Herzstillstand,
intrakranialer Hämorrhagie, Subarachnoidalblutung und intrakranielle Läsionen, Läsionen
des Rückenmarks, intravertebrale Läsionen, Schütteldrauma, infektiöser Enzephalitis,
Meningitis und Kopfschmerzen besteht.
16. Verwendung nach Anspruch 14, wobei die chronische neurodegenerative und/oder neuroinflammatorische
Störung aus der Gruppe ausgewählt wird, die aus Demenzen, Pick-Krankheit, diffuser
Lewy-Körper-Krankheit, progressiver supranukleärer Lähmung (Steel-Richardson-Syndrom),
Multipler Sklerose, Multipler System Atrophie, chronischer epileptischen Bedingungen
verbunden mit Neurodegeneration, Motoneuronen-Erkrankungen, degenerativen Ataxien,
kortikaler basale Degeneration, ALS-Parkinson-Demenz-Komplex von Guam, subakuter sklerosierende
Panenzephalitis, Huntington-Krankheit, Parkinson-Krankheit, Synucleinopathien, primärer
progressive Aphasie, striatonigraler Degeneration Machado-Joseph-Krankheit/spinozerebellarer
Ataxie Typ 3 und olivopontozerebellarer Degenerationen, Gilles de la Tourette-Krankheit,
Bulbär- und Pseudobulbärparalyse, Wirbelsäulen- und spinobulbärer Muskelatrophie (Kennedy-Krankheit),
primärer Lateralsklerose, familiärer spastische Paraplegie, Werdnig-Hoffmann-Krankheit,
Kugelberg-Welander Krankheit, Tay-Sachs-Krankheit, Sandhoff Krankheit, familiärer
spastischer Krankheit, spastischer Paraparese, progressiver multifokale Leukenzephalopathie,
familiärer Dysautonomie (Riley-Day-Syndrom), Polyneuropathie, Prionen-Erkrankungen,
Sucht, affektive Störungen, schizophrene Störungen, chronischem Erschöpfungssyndrom
und chronische Schmerzen besteht.
17. Verwendung nach den Ansprüchen 13 oder 14, wobei der Zustand Altern ist.
18. Verwendung nach den Ansprüchen 13 oder 14, wobei das Medikament vor oder nach dem
Einsetzen der Erkrankung verabreicht wird.
1. Variante de l'érythropoïétine (EPO) codant pour un polynucléotide sélectionné parmi
le groupe constitué :
(a) de polynucléotides codant pour la forme mature des polypeptides nommés hs3, h1-4,
h1-5, hs4, h1-1, h2-1, mS, mG3, mG5, m301, mK3, ha, hAma, hAmE, et hA-10 ayant la
séquence d'acides aminés déduite telle qu'illustrée dans SEQ ID N° : 2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 50, 51, 52, et 53, respectivement ;
(b) d'un polynucléotide codant pour la forme mature du polypeptide nommé séquence
ha sans séquence de tête constitué de la séquence d'acides aminés déduite telle qu'illustrée
dans SEQ ID N° : 61 ;
(c) de polynucléotides ayant la séquence codante, telle qu'illustrée dans SEQ ID N°
: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 55, 56, 57, et 58 codant pour au moins la
forme mature du polypeptide ;
(d) d'un polynucléotide constitué de la séquence codante, telle qu'illustrée dans
SEQ ID N° : 60 codant pour la forme mature du polypeptide nommé séquence ha sans séquence
de tête ;
(e) d'un polynucléotide codant pour une version humanisée des polypeptides mS, mG3,
mG5, m301 et mK3 constitués de la séquence d'acides aminés déduite telle qu'illustrée
dans SEQ ID N° : 14, 16, 18, 20, et 22 ;
(f) de polynucléotides codant pour un polypeptide comprenant une fusion d'une séquence
d'acides aminés sélectionnée parmi le groupe de séquences d'acides aminés telles qu'illustrées
dans SEQ ID N° : 24, 26, 28, et 30, au niveau de l'extrémité N d'une séquence d'acides
aminés sélectionnée parmi le groupe de séquences d'acides aminés telles qu'illustrées
dans SEQ ID N° : 32, 34, 36, et 38, dans lesquelles ladite fusion a une activité protectrice,
et en particulier neuroprotectrice, des cellules mais essentiellement aucune activité
hématopoïétique ;
(g) de polynucléotides comprenant une fusion de séquences polynucléotidiques sélectionnées
parmi le groupe de séquences polynucléotidiques telles qu'illustrées dans SEQ ID N°
: 23, 25, 27, et 29, 5' de la séquence polynucléotidique sélectionnée parmi le groupe
de séquences polynucléotidiques telles qu'illustrées dans SEQ ID N° : 31, 33, 35,
et 37, dans lesquelles ladite fusion a une activité protectrice, et en particulier
neuroprotectrice, des cellules mais essentiellement aucune activité hématopoïétique
;
(h) de polynucléotides codant pour un dérivé d'un polypeptide codé par un polynucléotide
selon l'un quelconque des points (a) à (g), dans lesquels dans ledit dérivé entre
1 et 10 résidus d'acides aminés sont substitués de façon conservatrice par rapport
au dit polypeptide, et ledit dérivé a une activité protectrice, et en particulier
neuroprotectrice, des cellules mais essentiellement aucune activité hématopoïétique
;
(i) de polynucléotides codant pour un fragment de n polypeptide codé par un polynucléotide
selon l'un quelconque des points (a) à (h), dans lesquels dans ledit fragment entre
1 et 10 résidus d'acides aminés subissent une délétion N et/ou C-terminale et/ou entre
1 et 10 acides aminés subissent une délétion N et/ou C-terminale de la jonction par
rapport au dit polypeptide, et ledit fragment a une activité protectrice, et en particulier
neuroprotectrice, des cellules mais essentiellement aucune activité hématopoïétique
;
(j) de polynucléotides qui sont identiques à au moins 95 % à un polynucléotide tel
que défini dans l'un quelconque des points (a) à (d) et qui en même temps ont une
activité protectrice, et en particulier neuroprotectrice, des cellules mais essentiellement
aucune activité hématopoïétique ;
(k) de polynucléotides dont le brin complémentaire s'hybride dans des conditions strictes
à un polynucléotide tel que défini dans l'un quelconque des points (a) à (j) et qui
codent pour un polypeptide ayant une activité protectrice, et en particulier neuroprotectrice,
des cellules mais essentiellement aucune activité hématopoïétique ;
(l) de polynucléotides codant pour un polypeptide de variante de l'EPO, qui comprend
la partie N-terminale de l'EPO de longueur totale incluant l'hélice A et à qui il
manque au moins un des suivants :
(i) un fragment d'au moins 10 acides aminés entre l'hélice A et l'hélice B ;
(ii) un fragment d'au moins 10 acides aminés de l'hélice B ;
(iii)un fragment d'au moins 6 acides aminés entre l'hélice B et l'hélice C ;
(iv) un fragment d'au moins 10 acides aminés de l'hélice C ;
(v) un fragment d'au moins 20 acides aminés entre l'hélice C et l'hélice D ; et/ou
(vi) un fragment d'au moins 10 acides aminés de l'hélice D ;
dans lesquels ladite variante a une activité protectrice, et en particulier neuroprotectrice,
des cellules mais essentiellement aucune activité hématopoïétique ;
(m) de polynucléotides codant pour un dérivé d'un polypeptide codé par un polynucléotide
selon l'un quelconque de (1), dans lesquels dans ledit dérivé entre 1 et 10 résidus
d'acides aminés sont substitués de façon conservatrice par rapport au dit polypeptide,
et ledit dérivé a une activité protectrice, et en particulier neuroprotectrice, des
cellules mais essentiellement aucune activité hématopoïétique ;
(n) de polynucléotides dont le brin complémentaire s'hybride dans des conditions strictes
à un polynucléotide tel que défini dans l'un quelconque des points (1) à (m) et qui
codent pour un polypeptide ayant une activité protectrice, et en particulier neuroprotectrice,
des cellules mais essentiellement aucune activité hématopoïétique ;
ou le brin complémentaire d'un tel polynucléotide.
2. Polynucléotide selon la revendication 1, qui est un ADN, un ADN génomique ou un ARN.
3. Vecteur contenant le polynucléotide selon la revendication 1 ou la revendication 2.
4. Vecteur selon la revendication 3, dans lequel le polynucléotide est fonctionnellement
lié à des séquences témoins d'expression permettant l'expression dans des cellules
hôtes procaryotes et/ou eucaryotes.
5. Cellule hôte génétiquement mise au point avec le polynucléotide selon la revendication
1 ou la revendication 2 ou le vecteur selon la revendication 3 ou la revendication
4.
6. Mammifère non humain transgénique sélectionné parmi le groupe constitué de primate
non humain, cheval, bovin, mouton, chèvre, cochon, chien, chat, lapin, souris, rat,
cochon d'inde, hamster et gerbille comprenant le polynucléotide selon la revendication
1 ou la revendication 2, un vecteur selon la revendication 3 ou la revendication 4
et/ou une cellule hôte selon la revendication 5.
7. Processus de production d'un polypeptide de variante de l'EPO codé par le polynucléotide
selon la revendication 1 ou la revendication 2, comprenant les étapes consistant à
: mettre en culture la cellule hôte selon la revendication 5 et récupérer le polypeptide
codé par ledit polynucléotide.
8. Processus selon la revendication 7, comprenant en outre l'étape consistant à modifier
ladite variante de l'EPO, dans lequel la modification est sélectionnée parmi le groupe
constitué de l'oxydation, de la sulfatation et phosphorylation, de l'addition d'oligosaccharides
ou de combinaisons de celles-ci.
9. Processus de production de cellules capables d'exprimer au moins une des variantes
de l'EPO comprenant des cellules génétiquement mises au point in vitro avec le vecteur selon la revendication 3 ou la revendication 4, dans lequel ledit/lesdits
polypeptide(s) de variante de l'EPO est/sont codés par le polynucléotide selon la
revendication 1 ou la revendication 2.
10. Polypeptide ayant la séquence d'acides aminés codée par le polynucléotide selon la
revendication 1 ou la revendication 2 ou pouvant être obtenu par le processus selon
la revendication 7 ou la revendication 8.
11. Composition pharmaceutique comprenant le polynucléotide selon la revendication 1 ou
la revendication 2, un vecteur selon la revendication 3 ou la revendication 4, une
cellule hôte selon la revendication 5, et/ou un polypeptide selon la revendication
10 et un ou plusieurs supports pharmaceutiquement acceptables.
12. Utilisation du polynucléotide selon la revendication 1 ou la revendication 2, d'un
vecteur selon la revendication 3 ou la revendication 4, d'une cellule hôte selon la
revendication 5, ou d'un polypeptide selon la revendication 10, pour la fabrication
d'un médicament pour le traitement ou la prévention d'une condition associée à l'endommagement
tissulaire dû à l'apoptose.
13. Utilisation selon la revendication 12, dans laquelle ladite apoptose est induite par
une ischémie, une hypoxie, une infection bactérienne, une infection virale, induite
de façon autoimmunologique, traumatique, chimique, ou induite par rayonnement.
14. Utilisation selon la revendication 12 ou la revendication 13, dans laquelle ladite
condition est un trouble neurodégénératif et/ou neuroinflammatoire aigu ou chronique,
est un trouble aigu ou chronique du coeur, du poumon, du rein, du foie ou du pancréas
ou ladite condition est associée à une greffe d'organe ou de cellules.
15. Utilisation selon la revendication 14, dans laquelle ledit trouble neurodégénératif
et/ou neuroinflammatoire aigu est sélectionné parmi le groupe constitué de l'ischémie
cérébrale ou l'infarctus, y compris l'occlusion par embolie et l'occlusion par thrombose,
la reperfusion suivant une ischémie aiguë, une blessure hypoxique-ischémique périnatale,
un arrêt cardiaque, une hémorragie intracrânienne, une hémorragie sous-arachnoïdienne
et des lésions intracrâniennes, des lésions de la colonne vertébrale, des lésions
intravertébrales, une entorse bénigne du rachis, le syndrome du bébé secoué, une encéphalite
infectieuse, une méningite, et une céphalée.
16. Utilisation selon la revendication 14, dans laquelle ledit trouble neurodégénératif
et/ou neuroinflammatoire chronique est sélectionné parmi le groupe constitué de démences,
de la maladie de Pick, de la maladie à corps de Lewy diffuse, d'une paralysie supranucléaire
progressive (syndrome de Steel-Richardson), d'une sclérose en plaque, d'une atrophie
multisystématisée, de conditions épileptiques chroniques associées à la neurodégénérescence,
de maladies neuromotrices, d'ataxies dégénératives, d'une dégénérescence de la région
corticale ou basale, du complexe de l'ALS et de la démence de Parkinson de Guam, de
la panencéphalite subaiguë sclérosante, de la maladie de Huntington, de la maladie
de Parkinson, de synucléinopathies, d'aphasie progressive primaire, de dégénérescence
striatonigrique, de la maladie de Machado-Joseph/de l'ataxie spinocérébelleuse de
type 3 et de dégénérescences olivopontocérébelleuses, de la maladie de Gilles de la
Tourette, d'une paralysie bulbaire et pseudobulbaire, d'une atrophie musculaire spinale
et spino-bulbaire (maladie de Kennedy), d'une sclérose latérale primitive, de la paraplégie
spastique familiale, de la maladie de Werdnig-Hoffmann, de la maladie de Kugelberg-Welander,
de la maladie de Tay-Sach, de la maladie de Sandhoff, de la maladie spastique familiale,
de la paraparésie spastique, de la leucoencéphalopathie multifocale progressive, de
la dysautonomie familiale (syndrome de Riley-Day), de polyneuropathies, de maladies
au prion, de l'addiction, de troubles affectifs, de troubles schizophréniques, du
syndrome de fatigue chronique, et de la douleur chronique.
17. Utilisation selon la revendication 13 ou la revendication 14, dans laquelle ladite
condition est le vieillissement.
18. Utilisation selon la revendication 13 ou la revendication 14, dans laquelle le médicament
est administré avant ou après le déclenchement de ladite condition.